Bioactive sphingolipids in health and disease: lipidomic analysis, metabolism and roles in membrane signaling and autophagy.

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A. H. Merrill, Jr.
School of Biology, Chemistry and Biochemistry and the Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332-0230, USA.

Organisms usually contain large numbers of sphingolipid subspecies (for a pathway based compilation, see www.sphingomap.org) and knowledge about the types and amounts is imperative because they influence membrane structure, interactions with the extracellular matrix and neighboring cells, vesicular traffic and the formation of specialized structures such as phagosomes and autophagosomes, as well as participate in intracellular and extracellular signaling. Fortunately, “sphingolipidomic” analysis is becoming feasible for important subsets such as all of the backbone “signaling” subspecies (ceramides, ceramide 1-phosphates, sphingoid bases, sphingoid base 1-phosphates, inter alia) using mass spectrometry,1 and these profiles are revealing many surprises,2 such as that under certain conditions cells contain significant amounts of “unusual” species such as N-mono-, di-, and tri- methyl-sphingoid bases (including N,N-dimethylsphingosine) and dihydroceramides (DHCer), in the latter case, in very high proportions when cells are treated with the anticancer drug fenretinide (4- hydroxyphenylretinamide). The elevation of DHCer by fenretinide is befuddling because the 4,5-trans-double bond of Cer has been thought to be required for biological activity; however, DHCer induces autophagy and may be important in the regulation of this important cellular process. The complexity of the sphingolipidome is hard to imagine, but one hopes that, when partnered with other systems biology approaches, the causes and consequences of the complexity will explain how these intriguing compounds are involved in almost every aspect of cell behavior and the malfunctions of many diseases.
1Merrill et al., Methods 2005 36:207. 2Zheng et al., Biochim. Biophys. Acta 2006 in press.

Influence of Sphingolipids on Expression of the Multidrug Resistance Phenotype1,2.

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V. Gouaze-Andersson, A. J. Kreitenberg, A. E. Giuliano, M. C. Cabot.
Department of Experimental Therapeutics, John Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA 90404, USA.

Natural product anticancer agents enhance intracellular levels of ceramide, a sphingolipid that promotes cell apoptosis. However, multidrug-resistant (MDR) cancer cells may avoid the cytotoxic effects of chemotherapy by glycosylating ceramide. In the present study we examined the relationship between ceramide, ceramide metabolites and expression of the MDR1 gene in human breast cancer cell lines. MDR1 codes for P-glycoprotein (P-gp), a member of the ABC transporter superfamily of proteins that promote cellular efflux of chemotherapeutic agents.
Four wild-type (drug-sensitive) human breast cancer cell lines (MCF-7, T47D,MDA-MB-231, MDA-MB-435) were used to evaluate the influence of acute and chronic exposure to ceramide and ceramide metabolites on MDR1 mRNA,P-gp, and resistance to chemotherapeutic agents. Acute exposure (<72 hours) to C8- ceramide (5 μg/ml culture medium), a cell-permeable analog of ceramide, enhanced MDR1 mRNA levels by 3- and 5-fold in T47D and inMDA-MB-435 cells, respectively, but did not affect MCF-7 cells.D-erythro-sphingosine exposure (5 μg/ml, 72 hr) increased MDR1 mRNA levels in MDA-MB-435 cells by 3.5-fold. Chronic exposure (> 72 hours) of MDA-MB-231cells to C8-ceramide enhanced MDR1 mRNA levels by 45- and 370-fold(real-time RT-PCR) at passages 12 and 22, respectively, and elicited expression of P-gp (Western blot). These cells not only demonstrated a 3-foldincrease in resistance to doxorubicin and a 9-fold increase in resistance to paclitaxel, but also showed increased efflux of rhodamine. Acute exposure ofMCF-7 and MDA-MB-231 cells to C8-glucosylceramide (10 μg/ml culture medium), a cell-permeable analog of glucosylceramide, increased MDR1 mRNA levels by 2- and 4-fold, respectively. Exposure of cells to either octanoic acid (C8:0), a C8-ceramide hydrolysis product, or oleic acid (C18:1) did not affect MDR1 expression.
These data are the first evidence that chronic exposure to ceramide and its metabolites enhances expression of the MDR phenotype in cancer cells. The impact of sphingolipids on MDR1 gene expression in cancer appears to reflect the myriad intracellular signaling pathways of ceramide and its metabolites. Although the mechanisms of gene activation are still unclear, ceramide’s role as a messenger of cytotoxic response to chemotherapy might be linked to the MDR pathway.
Supported by Susan B. Komen Breast Cancer Foundation; Department of Defense Breast Cancer Research Program; Associates for Breast and Prostate Cancer Studies, Los Angeles; and Fashion Footwear Association of New York Charitable Foundation (Shoes on Sale/QVC).
1Gouaze et al., Mol. Cancer Ther. 2004 3:633. 2Gouaze et al., Cancer Res. 2005 65:3861.

Balancing (grants) between peroxisomal and sphingolipid metabolism.

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Paul P. Van Veldhoven.
Farmakologie, Dept. Mol. Cell Biol., K.U.Leuven, Campus Gasthuisberg, Herestraat, B-3000 Leuven, Belgium.

Over the last three decades, the role of peroxisomes in mammalian lipid metabolism has been actively studied by our group. Although not obvious at first sight, a few links between peroxisomes and sphingolipid metabolism have been explored or discovered in recent years. A first one concerns the degradation of alpha-hydroxylated fatty acids (2-OH-FA), best known as constituents of certain sphingolipids (cerebrosides, ceramides). Our analysis of ceramide-phosphate in mouse tissues revealed also the presence of a2-OH- fatty N-acylated species in cerebellum. These 2-OH-FAs appear to be degraded by a modified alpha-oxidation pathway, involving an activation step and a cleavage reaction, generating formyl-CoA and a fatty aldehyde. The latter reaction is catalyzed by a peroxisomal enzyme that is also required for the removal of 3-methylbranched fatty acids (classical alpha-oxidation). In case of thiamine depletion or in peroxisome disorders, degradation of 2-OH-FAs is affected. Another example is related to C2-ceramide, the formation of which has been proposed to be dependent on platelet activating factor (PAF). Based on studies in a mouse model lacking peroxisomes, and unable to form (vinyl)etherlipids, such link seems highly questionable. Finally, given the fact that fatty aldehydes, produced during the last step of sphingolipid catabolism, are incorporated into (vinyl)etherlipids (plasmalogens), the responsible enzyme, sphingosine-phosphate lyase, was studied. This finally leads to the generation of a knock-out mouse, providing new insights insphingosine-1-phosphate mediated biology.

Cannabinoids and ceramide: two lipids acting hand by hand.

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M. Guzmán.
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Spain.

Cannabinoids, the active components of Cannabis sativa (marijuana) and their endogenous counterparts, exert their effects by binding to specific Gi/o-protein-coupled receptors that modulate adenylyl cyclase, ion channels andmitogen-activated protein kinases. Research carried out by our group during the last years has shown that cannabinoid receptors are also coupled to the generation of ceramide via two different pathways: sphingomyelin hydrolysis and ceramide synthesis de novo. Sustained ceramide accumulation in tumor cells mediates cannabinoid-induced apoptosis, as evidenced by in vitro andin vivo studies. Former experiments supported that this effect was due to the impact of ceramide on key cell signaling pathways such as the extracellularsignal-regulated kinase cascade and the Akt pathway. More recently we have identified a new route that mediates cannabinoid-induced apoptosis of tumor cells via de novo-synthesized ceramide. This route involves the stress- regulated protein p8 (also designated as candidate of metastasis 1) and its downstream targets ATF-4, CHOP and TRB3, which had been previously implicated in the endoplasmic reticulum stress response. These findings provide a new conceptual view on how cannabinoids act, and raise interesting pathophysiological questions.

Biological evaluation of sphingolipid analogues as potential antifungal agents.

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D. Mormeneo^1,2, J. Casas¹, A. Manresa³, A. Delgado^1,2.
¹RUBAM, Department of Biological Organic Chemistry, IIQAB-CSIC, C/ Jordi Girona 18-26, 08034 Barcelona and Units of ²Pharmaceutical Chemistry and3Microbiology, School of Pharmacy, University of Barcelona, Avda. Joan XXIII s/n; 08029 Barcelona, Spain.

Over the last 20 years, the number of patients with serious fungal opportunistic infections has been rising because of increased number of immune suppressed patients, either by infection with HIV, intense chemotherapy for treatment of cancer or organ transplantation. Unfortunately, the structural and metabolic similarities between fungal and mammalian cells have made the identification of novel antifungal targets extremely difficult. In this sense, differences between sphingolipid metabolism in fungi and mammals might lead to new pharmacological targets. The present work presents the design, synthesis and biological evaluation of phytosphingosine (PHS) and phytoceramide (PHC) analogues as fungal growth inhibitors.1 From a chemical standpoint, the new entities differ from the natural sphingoid bases in the nature of C1 position, side chain at C2 position and stereochemistry of C3 and C4 stereogenic centers (figure). All these analogues were screened preliminarily over Saccharomyces cerevisiaecultures and the most promising structures were evaluated over different yeasts and
The present work has been funded by an Education and Science Ministry fellowship (ref.AP2002-0567)moulds. In addition to these in vivo approaches, some of PHC analogues were also screened as in vitro inhibitors over the essential fungal enzyme inositolposphorylceramide synthase (IPCS).2
1Chung et al., J. Biol. Chem. 2001 276:35614. 2Georgopapadakou, Expert Opin. Investig. Drugs 2000 9:1787.

Synthesis of novel scyphostatin analogues and evaluation as neutral sphingomyelinase inhibitors.

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E. N. Pitsinos¹, A. Cruz¹, A. Giannis², V. Wascholowski².
¹Lab. of Natural Products Synthesis & Bioorganic Chemistry, Inst. of Physical Chemistry, NCSR “DEMOKRITOS”, P.O. Box 60228, 153 10 Ag. Paraskevi, Greece. ²University of Leipzig, Institute of Organic Chemistry, Johannisallee 29, 04103 Leipzig, Germany.

Sphingomyelinases are enzymes that, in response to specific stimuli, catalyse the hydrolysis of sphingomyelin to ceramide. Of the various isotypes described to date, the plasma membrane-located neutral sphingomyelinase (N-SMase) is of particular interest as a target for the development of new drugs for the treatment of neurodegenerative diseases, such as Alzheimer’s disease.1

We have recently developed a short, versatile and efficient strategy towards the pharmacophoric polar core of Scyphostatin.3 Modification of this strategy has allowed us to prepare several analogues of the natural product (2-3).Details of the synthetic route employed as well as their activity as N-SMaseinhibitors will be presented.4The natural product Scyphostatin (1) is a potent and selective inhibitor (IC50 = 1 μΜ) of N-SMase. However, its chemical stability is limited due to the lipophilic polyunsaturated side chain.2
1Wascholowski and Giannis, Drug News & Perspectives 2001 14:581. 2Yang et al., Neurobiology of Disease 2004 17, 99; Nara et al., J. Antibiot. 1999 52:525. 3Pitsinos and Cruz, Org. Lett. 2005 7:2245. 4Wascholowski et al., ChemMedChem 2006 1:718.

Phospholipid metabolism modulators in pathogenic organisms.

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P. González-Bulnes¹, J. Casas1, C. Ben Mamoun² and A. Llebaria¹.
¹Department of Organic Biological Chemistry (IIQAB-CSIC), Jordi Girona 18- 26, 08034 Barcelona, Spain. ²Department of Genetics and Developmental Biology (UCHC), 263 Farmington Avenue, Farmington CT 06030, USA.

Aminohydroxamic acids represent a new class of compounds which can affect phospholipid metabolism. The compounds presented here were originally designed as inhibitors of PC-PLCBc1, a phospholipase that catalyzes the hydrolysis of phosphatidylcholine to form DAG and phosphorylcholine. Study of their biological activity showed that they inhibited the targeted enzyme with IC50 values ranging between 4 and 100 µM. The similarity between the phospholipase of Bacillus cereus and that of Clostridium perfringens2, the bacterium which causes gas gangrene, suggested that the aforementioned compounds could also inhibit this enzyme. However, in vitro studies demonstrated that these compounds had no effect against the phospholipase C of Clostridium perfringens for most of the compounds. The fact that sphingomyelin synthase uses the same substrate than phospholipase C and catalyzes a similar reaction made us think that these compounds might inhibit sphingomyelin synthase. Interestingly all compounds were found to inhibit this enzyme albeit at a higher inhibitory concentration. Finally, the report of a phospholipase C as a good target for synthesis of antimalarial3 drugs made us think that these aminohydroxamic acids may inhibit Plasmodium falciparum, the parasite that causes malaria. A preeliminary screening made by the WHO showed that they inhibited the growth of Plasmodium falciparum as well as that of Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei rhodesiense with little or no cytotoxicity in mammalian cells. Because of their strong antimalarial activity, a more thorough study of the mode of action of these compounds in P. falciparum was performed. The structural similarity between them and miltefosine4 made us think that the effect against the parasite could be the same. Being miltefoxine a lipid inhibitor and affecting our compounds enzymes implicated in lipid metabolism the first thing to do was to assess the effect over lipid biosynthesis. The fact that the stage of P. falciparum life cycle inhibited by these compounds was the trophozoite stage, when the production of lipids increases to form the membranes of the merozoites, reinforced this idea. Labelling studies showed a dose-dependent inhibition of synthesis of phosphatidylcholine, the predominant phospholipid in the parasite’s membrane, and no effect on the synthesis of phosphatidylethanolamine. The IC50 values of these compounds were not affected by the availability of choline and were similar between wild type and transgenic parasites overexpressing or lacking an enzyme involved in the early steps of synthesis of phosphatidylcholine from serine. Together these studies suggest that aminohydroxamic acids exert their antimalarial activity via inhibition of the last essential steps of synthesis of phosphatidylcholine.
1González-Roura et al., Angew. Chem. Int. Ed. 2004 43:852. 2Naylor et al., Nat. Struc. Biol. 1998 5:738. 3Hanada et al., J. Exp. Med. 2002 195:23. 4Pessi et al., Proc. Natl. Acad. Sci. 2004, 101:6206.

Suppression of the gene expression of GD3 synthase in human melanoma cells blocks the ability of the tumor cells to induce neoangiogenesis upon xenografting in nude mice.

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I. Popa, S. Trompezinski, L. Thomas, J. Portoukalian.
University of Lyon-1, Dermatology, Edouard Herriot Hospital, 69437 Lyon Cx 03, France.

We established several years ago a model of human metastatic melanoma with immunosuppressed newborn rats by subcutaneously grafting a single human melanoma cell line. The lung metastases were repeatedly excised, grown in culture and grafted again to produce increasingly metastatic variants. After many cycles, a set of ten variants was obtained, each one showing a stable and reproducible metastatic potential that ranged from 1 to more than 300 lung metastases in the newborn rats. Along with the lung metastases, all variants gave a tumor at the s.c. injection site in three weeks following grafting of one million cells. The analysis of gangliosides of the variants showed a highly significant reverse correlation between the GD3 ganglioside content and the metastatic potential.1 The highly metastatic variants had a strongly reduced ganglioside content with a nearly complete absence of GD3, a much lower amount of GM3 and a strong increase in lactosylceramide as compared to the poorly metastatic ones. However, one striking observation was that keeping in culture the highly metastatic variant TW12 over a ten passage period progressively brought back its ganglioside pattern to that of the non-metastatic one IC8 (containing high amounts of both GM3 and GD3), along with a loss of its metastatic potential. These changes of the ganglioside pattern were also seen in vivo since all the tumors grown at the s.c. injection site in rats had the ganglioside pattern of the original non-metastatic variant, regardless of their lung metastatic potential, even when cloned cells were injected.2 The possible role of GD3 synthase gene expression in the formation of melanoma metastases was studied after transfection of the non-metastatic variant IC8 with plasmids containing antisense sequences for GD3 synthase. Highly metastatic IP-1 cells were selected from GD3 antisense-transfected IC8 cells using Matrigel-coatedBoyden chambers. The cells that crossed the chambers were expanded and the sphingolipid pattern was found to match exactly the one of the highly metastatic one TW12. Neo-angiogenesis is critical for the growth of metastases and the role of GD3 was investigated in vitro with human vein endothelial cells (HUVEC) and in vivo with Matrigel-embedded melanoma cells injected in nude mice. The results showed that exogenously added GD3 strongly increases in vitro angiogenesis. Moreover, blocking the GD3 synthase gene expression in IP-1 cells nearly abolished angiogenesis in nude mice, whereas IC8 induced a strong angiogenesis in a two-week period. Interestingly, TW12 embedded in Matrigel and injected to the nude mice expressed GD3 and induced angiogenesis, whereas GD3 antisense-transfectedTW12 did not, supporting the hypothesis that GD3 may be produced again by proliferating highly metastatic cells to ensure a proper neo-angiogenesis.
1Thomas et al., C. R. Acad. Sci. III. 1995, 318:1233. 2Zebda et al., FEBS Lett. 1995, 362:161.

Developmental expression of the sialidase Neu2 ortholog gene in zebrafish and characterization of the knock-down model.

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A. Fanzani¹, F. Colombo¹, S. Nicoli², R. Giuliani¹, D. Zizioli¹, F. Cotelli³, S. Rossi¹, D. Maiolo¹, M. Presta², A. Preti¹, S. Marchesini¹.
Department of Biomedical Sciences and Biotechnology, Unit of ¹Biochemistry and ²Pathology, University of Brescia, Italy and ³Department of Biology, University of Milan, Italy.

Neu2 is a soluble sialidase that has been suggested to play an important role in skeletal muscle differentiation. However, the role of Neu2 gene during embryogenesis has not been ever investigated. In the present study, in silico analysis identified in zebrafish genome six genes encoding sialidase-likeproteins. Here we reported the cloning of cytosolic sialidase Neu2 ortholog in zebrafish. The Neu2 gene is located on chromosome 21 with 3 ORF exons encoding a 376 amino acid protein which has 57-59% homology to mammalian Neu2 forms. This protein contains all the conserved sialidase amino acid motifs, and the enzyme heterologously expressed in mammalian cells is present in the cytosol and it is functional toward the artificial substrate 4-MU-NeuAc in a range of neutral pH typical of soluble sialidases. During zebrafish embryogenesis, Neu2 resulted strongly expressed in the early stages along the somitogenesis, while the expression after 24-48 hours post fertilization resulted in the anterior and caudal regions of the embryos. The knock-downmodel of Neu2 gene obtained by morpholinos microinjection revealed a severe phenotype, underlying an emerging role of this enzyme during vertebrate development.

Ceramide kinase in inflammation: What do we learn from KO mice?

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S. Niwa, B. Zemann, C. Graf, P. Rovina, R. Reuschel, D. Mechtcheriakova, N. Urtz, B. Kinzel, M. Müller, T. Baumruker, A. Billich, F. Bornancin.
Novartis Institutes for BioMedical Research, Vienna, Austria, and Basel, Switzerland.

Ceramide kinase (CerK) uses ATP to phosphorylate ceramide, leading toceramide-1-phosphate (C1P). CerK/C1P have been recognized as new players in inflammation, controlling mechanisms such as phagocytosis, mast cell degranulation and cPLA2 activation. We generated CerK-/- mice by homologous recombination in Balb/C background. The resulting animals are viable, breed normally, and do not display any overt phenotype. By using in vitro as well as in vivo models, we have started investigating the dependency of critical inflammatory responses on the presence of CerK. The results of these ongoing studies will be presented.

Construction of a conditional knockout mouse model of the ceramide- kinase like gene (CERKL).

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A. Garanto, M. Tuson, R. González-Duarte, G. Marfany.
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028-Barcelona, Spain.

Retinitis pigmentosa (RP) is the main cause of blindness in human, affecting1:3000-1:5000 adults. RP defines a clinically and genetically group of retinal disorders caused by progressive photoreceptor neurodegeneration and for which there is not any effective therapy yet. Mutations in at least 32 genes have been associated to this disorder and more than 18 genes are responsible for the autosomal recessive forms (arRP). Recently, our group identified a novel causative gene, CERKL (ceramide kinase-like gene), in three consanguineous pedigrees from Spain. Remarkably, the mutation introduced a STOP codon within the lipid kinase domain, generating a prematurely truncated protein. Although sequence homology clearly involves the CERKL protein in the sphingolipid metabolism, its substrate(s) and precise physiological function remain as yet unknown. Ceramide has already been shown to be a key regulator of retinal cell apoptosis. We surmise that CERKL activity may protect the cell against ceramide production in stressed retinal cells. Therefore, this mutation of CERKL would preclude the phosphorylation of ceramide, triggering photoreceptor cell death and eventually causing blindness.
Although natural mouse mutants that serve as models for retinal diseases have been collected since the beginning of the XXth century, very few of them are good models for retinitis pigmentosa. Besides, given the high number of genes involved in RP, the available knockout models are also very scarce. In order to shed light on the biological function of CERKL as well as on the pathogenic mechanisms by which the disfunction of the sphingolipid metabolism causes retinitis, we designed a conditional knockout mouse model (cre-loxP) to disrupt the CERKL gene. Last but not least, this knockout mouse will provide a model in which to validate the effectiveness of new therapeutic approaches.

Lipid rafts rich in sphingomyelin and cholesterol content in the nucleus: a platform for transcription process.

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G. Cascianelli, M. Villani, M. Tosti, F. Marini, M. P. Viola Magni, E. Albi
Department of Clinical and Experimental Medicine, Physiopathology, University of Perugia, Italy.

The role of cholesterol (CHO) and sphingolipid in the generation of lipid raft domains in cell membrane has been widely described1. A relation between sphingomyelin (SM) and cholesterol (CHO) regulated by neutral sphingomyelinase (N-SMase) activity has been shown inside the nucleus2. The CHO SM-linked fraction changed during cell proliferation. The aim of the work was to ascertain first the existence of lipid microdomains in the nucleus. The microdomains were extracted from purified nuclei using TritonX-100 detergent at low temperature and were isolated by sucrose density gradient. The results showed the existence of nuclear lipid rafts which had a morphology similar to that previously reported for microvillar membrane. The biochemical analysia showed that nuclear microdomains were characterized by the presence of CHO, SM and phosphatidylcholine (PC) in the 1:1:1 ratio. The SM metabolism enzyme assay demonstrated the N-SMase,SM-synthase and reverse-SM-synthase activity suggesting that the SM was metabolized directly in lipid rafts probably in relation to cell function. To study the possible involvement in nuclear processes, we have analyzed the incorporation of radioactive uridine at 24 hours after partial hepatectomy which resulted very high in the nuclear lipid rafts. The presence of lamin B as a specific protein of nuclear membrane and STAT3 transcription factor suggested that the nuclear lipid rafts could represent a platform for transcription process in nuclear membrane.
1Edidin M. Ann. Rev. Biophys. Biomol. Struct. 2003 32: 257¸ 2Albi E and Viola Magni MP. J. Hepatol. 2002 36: 395

Modulation of sphingolipid metabolism alters vascular responses to angiotensin II and muscarinic receptor agonists.

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A. C. M. Mulders, M. C. Hendriks-Balk, M.-J. Mathy, M. C. Michel, A. E. Alewijnse, S. L. M. Peters.
Dept. Pharmacology and Pharmacotherapy, Academic Medical Center, Amsterdam, The Netherlands.

Sphingolipids are major constituents of the cellular membrane and play important roles in maintaining cellular homeostasis. The sphingolipid metabolites sphingomyelin, ceramide, sphingosine and sphingosine-1-phosphate can be metabolized into eachother and can have opposite effects on cell growth and survival. It has been suggested for programmed cell death, cell survival and cell growth that it is not the absolute amount, but the relative balance between these sphingolipid metabolites which is decisive. Next to this, sphingolipids also have vasoactive properties, and can induce vascular relaxation or constriction depending, amongst others, on vessel type. Besides their occurrence in plasma, they can also be formed locally in the vascular wall itself in response to external stimuli, for example TNFα. We investigated whether the known vasoactive compounds angiotensin II and methacholine modulate the sphingolipid metabolism in the vascular wall and how this might contribute to the vascular responses.
In isolated rat carotid arteries, the contractile responses to angiotensin II were enhanced by the sphingosine kinase inhibitor N,N dimethylsphingosine (DMS, 10 µM). Endothelium removal or NO synthase inhibition withnω-nitro-L- arginine (L-NNA, 100 µM) resulted in a similar enhancement. Applied together, DMS had no additional effect over the L-NNA-induced shift of the concentration response curve. Angiotensin IIconcentration-dependently induced NO production in an endothelial cell line, which could be diminished by DMS. Using immunoblotting and intracellular calcium measurements, we demonstrated that this sphingosinekinase-dependent endothelial NO synthase activation was mediated via both phosphatidylinositol 3-kinase/Akt and calcium-dependent pathways.
In translocation experiments in endothelial cells we showed that activation of the muscarinic M3 receptor lead to translocation of YFP-labelled sphingosine kinase from the cytoplasm to the cellular membrane. In relaxation experiments, inhibition of sphingosine kinase reduced the potency of relaxation response to the muscarinic receptor agonist methacholine in isolated rat carotid arteries and aorta. The endogenously formed S1P had a relaxant effect, in analogy to the angiotensin II pathway we have shown. Interestingly, in contrast to conduit vessels, DMS induced a significant enhancement of methacholine-induced relaxation in mesenteric artery preparations. This suggests that the endogenously formed S1P in this resistance vessel is a contractile factor in muscarinic M3-induced vascular relaxation.
We conclude that modulation of sphingolipid metabolism by vasoactive compounds such as angiotensin II and muscarinic receptor agonists, contributes to their vasoactive properties. For the muscarinic M3 receptor, this modulation of sphingolipid metabolism leads to differential effects in conduit and resistance vessels. The involvement of endogenous sphingolipid formation may be of importance under pathological circumstances with reduced NO bioavailability. Moreover, a disturbed sphingolipid metabolism in the vascular wall may lead to reduced NO bioavailability and endothelial dysfunction.

Involvement of the endoplasmic-reticulum during serum-withdrawal dependent apoptosis in hippocampal neuroblasts.

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V. Voccoli,¹ F. Mazzoni,¹ L. Colombaioni,¹ M. García-Gil,^2
1Istituto di Neuroscienze CNR, Via G.Moruzzi 1, 56100 Pisa, Italy and ²Dipartimento di Biologia, Unità di Fisiologia, Università di Pisa, Via S. Zeno 31, 56127 Pisa, Italy.

Apoptotic death caused by diseases or toxic insults is preceded and determined by endoplasmic reticulum dysfunction and altered intraluminar calcium homeostasis in many different cell types. With the present study we have explored the possibility that the ER stress could be involved also in apoptotic death induced by serum deprivation in neuronal cells. We have previously shown that in the embryonic hippocampal cell line HN9, serum deprivation induces nuclear sphingomyelinase activation together withsphingomyelin-synthase inhibition and a consequent increase of nuclear ceramide pool; this is followed by relocation of cytochrome c to cytosol and of Bax to mitochondria, deregulation of calcium metabolism without loss of mitochondrial functionality, and activation of caspase-3. The Ca++concentration in the lumen of the ER has been evaluated by using the low affinity Ca++ probe Mag-fluo-4. We show that serum deprivation lowers the ER Ca++ concentration with a time course closely related to the increase of apoptosis incidence. Serum deprivation also enhances the expression of a well known marker of ER stress, the glucose regulated protein-78 (GRP-78), a member of the heat shock/stress response protein family. Moreover, in serum deprived neuroblasts, following GRP-78 up-regulation, the ER-associatedprocaspase-12 is cleaved with a time course which parallels the ER calcium loss. These findings indicate that, in hippocampal neuroblasts, Ca++mobilization from ER and caspase-12 activation are components of the molecular pathway that leads to apoptosis triggered by serum deprivation and may constitute an amplifying loop of the mitochondrial pathway.

Sphingolipid modulation of immune cell function.

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E. A. Martinova.
State Institute of Nutrition Russian Academy of Medical Sciences, Moscow, Russian Federation.

Simple sphingolipids such as sphingosine, sphinganine, C2-ceramide as well as inhibitor of ceramide synthase – fumonisin B1 were found to modulate many signaling pathways into cell. This allows them to regulate the immune cell function. Previously we have shown that exogenously added sphingolipids changed the receptor expression in immunological synapse including CD3, CD4, CD8, CD45, CD54 and some others.1 This causes the disruption in T- helper cell differentiation and prevalence Th2 cells on Th1 cells.2 As a result the primary immune response to T-dependent antigens can be modulated by exogenous sphingolipids. The most effective lipid from mentioned above is sphingosine. D-sphinganine is less effective compared with D,L-sphinganinewhich is more toxic for immune cells and different cell lines. Sphingolipids activate the macrophage function via elevation of receptor F4/80 expression and chemokine production. The excellent tolls for immune cell regulation are three fumonisins – B1, B2, and B3 which may elevate or impair immune response dependent on its percentages in the mixture. Fumonisin B1 regulates the memory cell formation and secondary immune response toT-dependent antigens.3 Fumonisin B1 inhibits the DNA synthesis in normal lymphocytes in response to mitogenes, more effective in T cells compared with B cells.1 Under condition of fumonisin exposure it causes the prolonged immune deficiency and activation of growth of transformed cells. Effects of sphingolipids on immune cells are strong connected with cell cycle.4 It is not possible to initiate an apoptosis in some conditions in the synchronized cultured cells. In the absence of specific antigen sphingolipids lead to the activation induced apoptosis of lymphocytes. The practical implication of this one is an induction of apoptosis in the tumor-associated lymphocytes which lost the! normal anti-tumor defense. Endogenous sphingolipids participate as si gnaling molecules in all types of apoptosis such as receptor-dependent,mitochondrial-connected or stress-initiated. We also found that cell death after sphingolipid exposure may be rather by necrosis but not apoptosis. It may be under several conditions including additional activation of TNF signaling pathways4 or ATP depletion. We found the cross-talk betweensphingolipid-derived signaling and mitochondrial respiration. Inhibitors of respiration may modulate the sphingolipid initiated apoptosis in different cell types. One of mechanisms of fumonisin effect on lymphocytes is an activation of the neutral sphingomyelinase of plasma membrane and generation of sphingomyeline cycle.5 Our last data discovered the new mechanism of sphingolipid effect on immune cells. We found that exogenous added sphingosine, sphinganine or fumonisin B1 activated the stress-dependentsignaling pathways in endoplasmic reticulum followed by the expression of some mitochondrial proteins including Lon protease. This causes an activation of ATP-dependent proteolysis and perturbation of regulatory proteins data in press). Under stimulation of normal immune response, Lon protease expression is activated simultaneously with proteolysis of regulatory proteins. Simultaneous exposure of sphingolipids and inhibitors of Lon protease expression amplifies the apoptotic signals into immune cells. Conclusion: exogenous sphingosine, sphinganine, C2-ceramide and fumonisin affect some stages of immune cell differentiation, proliferation, and apoptosis followed by modulation of immune response.
1Martinova, Adv. Exp. Med. Biol. 1996 391:331. 2Martinova, Toxicon. 1997 35:497. 3Martinova and Merrill, Mycopathologia 1995 130:163. 4Martinova et al., Biomed. Khim. 2003 49:35.5Martinova et al., Biochemistry (Moscow) 1995 60:461.

The tricyclodecan-9-yl-xanthogenate D609 triggers ceramide increase and enhances FasL-induced caspase-dependent and -independent cell death in T lymphocytes.

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D. Milhas, N. Therville, T. Levade, H. Benoist*, B. Segui*
INSERM U466, IFR31 BP 84225 31432 Toulouse cedex 4 FRANCE.

D609 is known to modulate death receptor-induced ceramide generation and cell death. We show that in Jurkat cells, non-toxic D609 concentrations inhibit sphingomyelin synthase and, to a lesser extent, glucosylceramide synthase, and transiently increase intracellular ceramide level. D609 significantly enhanced FasL-induced caspase activation and apoptosis. D609 stimulated FasL-induced cell death in caspase-8-deficient Jurkat cells indicating that D609 acts downstream of caspase-8. At high FasL concentration (500 ng/mL), cell death was significantly, but not completely, inhibited byzVAD-fmk, a broad-spectrum caspase inhibitor, indicating that FasL can activate both caspase-dependent and -independent cell death signaling pathways. FasL- induced caspase activation was abolished by zVAD-fmkwhereas ceramide production was only partially impaired. D609 enhancedcaspase-independent ceramide increase and cell death in response to FasL. Also, D609 overcame zVAD-fmk-conferred resistance to a FasL concentration as low as 50 ng/mL and bypassed Rip deficiency. Mitochondrial events were likely involved since Bcl-xL over-expression impaired D609 effects. InPHA-activated human T lymphocytes, D609 enhanced FasL-induced cell death in the presence or absence of zVAD-fmk. Altogether, our data strongly indicate that the inhibition of ceramide conversion to complex sphingolipids by D609 is accompanied by an enhancement of FasL-inducedcaspase-dependent and – independent cell death in T lymphocytes.

Ceramide Mediates Amyloid Beta- and Tumor Necrosis Factor Alpha- Induced NADPH Oxidase Activation and Subsequent Oxidation of the Neuronal Actin Cytoskeleton.

O13
B. M. Barth^1,2, D. L. LaVictorie¹, S. J. Brown¹, C. M. McGill¹, T. P. Clausen¹, T. B. Kuhn^1,2.
¹Department of Chemistry and Biochemistry and ²Alaska Basic Neuroscience Program, University of Alaska-Fairbanks, Fairbanks, AK 99775 USA.

Inflammation and its mediators are implicated in the orchestration and progression of acute and chronic degenerative pathologies of the central nervous system (CNS). In particular, oxidative stress plays a major role in the progressive nature of CNS inflammation. Additionally, dysfunction of the neuronal actin cytoskeleton represents a crucial step in many degenerative CNS pathologies. The integrity and dynamics of the neuronal actin cytoskeleton is pivotal for growth cone motility, axon outgrowth and regeneration, and dendritic spine dynamics. In this study, we explored neurodegenerative mechanisms in SH-SY5Y human neuroblastoma cells exposed to either the proinflammatory cytokine tumor necrosis factor alpha (TNFα), or the amyloid beta fragment (Aβ) associated with Alzheimer’s pathology. We elucidated a molecular pathway linking TNFα or Aβ-exposureto the destruction of the neuronal actin cytoskeleton and dysfunction of neuronal motility processes. TNFα and Aβ both stimulated a rapid increase in ceramide by activating a neutral sphingomyelinase. The role of the neutral sphingomyelinase was evaluated pharmacologically, and by assay of enzymatic activity. Increases in ceramide further resulted in an activation of a neuronal NADPH oxidase (NOX) activity as revealed by phosphorylation and membrane translocation of cytosolic NOX components, and the production of oxygen radicals as revealed by fluorescent imaging. Lastly, 2,4- dinitrophenylhydrazine derivatization of protein carbonyls revealed irreversible oxidative damage to immuno-precipitated actin.
Together, our results implicate ceramide as a key intermediate of Aβ- andTNFα-induced NOX activation in neuronal cells. Moreover, this investigation links neuronal NOX activation with an irreversible oxidative damage to the neuronal actin cytoskeleton. Thus, inflammatory processes associated with acute and chronic CNS degeneration could directly contribute to the failure of axon regeneration. A more detailed understanding of this pathway in primary neurons could allow for the development of more specific therapeutic approaches to improve axonal regeneration and neuronal connectivity.
Supported by USDA grant number 2005-34495-16519 and NIH grant number U54 NS41069.

Ceramide regulates free calcium levels in endoplasmic reticulum of hippocampal neuroblasts: possible role in initiation of neuronal differentiation.

O14
V. Voccoli, L. Colombaioni.
Istituto di Neuroscienze, CNR. Via G. Moruzzi, 1 56100 Pisa, Italy.

Ceramide is largely known as a lipid second messenger capable of regulating different cellular functions. Increases in intracellular ceramide levels have been mainly related to the apoptosis onset, but also the processes of growth suppression or terminal differentiation are potentially controlled by this lipid or by its metabolites, such as ceramide-1-phosphate (C1P) or glucosylceramide (GlcCer). With this study we have analyzed the cellular signaling correlated to the early phases of neuronal differentiation in the hippocampal cell line HN9.10e. We have found that, in response to pro- differentiating stimuli, endoplasmic reticulum (ER) undergoes functional modifications that are closely correlated to the progression of the neuritic growth. In particular, the enhancement of ER free calcium, appears to be a key factor in promoting differentiation. We have observed that also the application of C2Cer, a membrane-permeant analogous of ceramide, at low doses (100-500nM) rapidly stimulates neuritic growth. C2Cer induces a free calcium increase in ER comparable to that observed during differentiation induced by agents such as retinoic acid (RA). Also the treatment with the ceramidase inhibitor N-oleoylethanolamine (NOE) at low doses (5 uM) and for short periods (2-5 hours) mimics the pro-differentiating effects of C2Cer, and produces a free calcium increase in ER. This indicates that also endogenous ceramide is capable of controlling the early phases of differentiation. We have previously demonstrated that 25-75 uM C2Cer has a pro-apoptotic action in HN9.10e neuroblasts. These data suggest that while relatively high levels of intracellular ceramide can trigger the cellular apoptotic death, lower levels are inductors of neuritic growth and differentiation. A direct action on calcium homeostasis in ER appears to be the major event in the intracellular signalling triggered by ceramide. It is possible that the observed effects on ER calcium homeostasis are not directly due to ceramide, but rather to some ceramide metabolite.

Metabolic and functional role of the ceramide binding protein CERT in glioma cells.

O15
P. Giussani¹, T. Colleoni¹, R. Bassi¹, L. Brioschi¹, G. Tettamanti¹, K. Hanada², L. Riboni¹, P. Viani¹.
¹Department of Medical Chemistry, Biochemistry and Biotechnology, Faculty of Medicine, University of Milan, Milan, Italy. ²Department of Biochemistry and Cell Biology, National Institute of Infection Diseases, Tokyo, Japan.

Different studies demonstrate that in glial cells ceramide (Cer) exerts antiproliferative and proapoptotic effects, and strongly support that Cer- signalling is altered in glial tumors. The control of ceramide levels in glial cells involves specific enzymes which are known to be localized in different subcellular compartments as well as Cer movements along the sphingomyelin biosynthetic pathway. A key element in defining the role of Cer in sphingolipid metabolism and signalling is its hydrophobic nature, and its consequent inability to spontaneously move among different subcellular sites where the enzymes of its metabolism and its molecular targets are located. Based on this evidence, the biological effect exerted by Cer may depend on the presence of specific signalling pools of this bioactive sphingoid in the cell, as well as the regulation of its intracellular traffic. Recently, the identification in CHO cells of the Cer specific carrier protein CERT have revealed a novel pathway for the delivery of Cer to the Golgi apparatus for sphingomyelin biosynthesis. In this study we investigated the metabolic and functional role of CERT in glioma cells. All glioma cells analyzed constitutively express CERT, the protein being mainly associated to the cytosolic fraction. Metabolic experiments performed with different radioactive metabolic precursors of sphigolipids indicate that, in all analyzed glioma cells, downregulation of CERT by RNA interference technology promoted a significant but not complete reduction of the amount of Cer converted to SM. This suggests that in glioma cells CERT mediated Cer transport contributes in addressing ceramide toward sphingomyelin biosynthesis. Since the regulation of sphingomyelin biosynthesis represents a crucial step in the role of ceramide on glial cell proliferation we evaluated the possible role of CERT in the control of glioma cell proliferation. We found that in all glioma cells down regulation of CERT resulted in an increased cell proliferation associated to higher ERK1/2 phosphorylation. In vitro and in vivo experiments indicated that CERT is necessary for the inhibition exerted by Cer on ERK activation. In conclusion these results suggest that CERT, besides the involvement in sphingolipid metabolism, participates to Cer signalling and could play a role in the control of glioma cell proliferation.

Ceramide 1- phosphate stimulates cell proliferation in bone marrow- derived macrophages through activation of the PI3-K/PKB (Akt) and MAPK pathways.

O16
P. Gangoiti¹, M. Granado¹, J. Y. Kong², U. P. Steinbrecher², A. Gómez- Muñoz¹.
¹Department of Biochemistry and Molecular Biology, School of Science and Technology, University of the Basque Country, 48080 Bilbao, Spain. ²Department of Medicine, University of British Columbia, Vancouver, Canada.

We previously reported that ceramide 1-phosphate (Cer 1-P) was bioactive as it stimulated DNA synthesis and cell division in rat or mouse fibroblasts, and prevented cells from entering apoptosis. A major mechanism whereby Cer 1-Pblocks apoptosis involves the inhibition of acid sphingomyelinase. However, the mechanisms or signaling pathways that are implicated in the mitogenic effect of Cer 1-P are incompletely understood.
In the present work, we show that Cer 1-P stimulates DNA synthesis and cell proliferation in bone marrow-derived macrophages. This effect was independent of phospholipase C or phospholipase D activation, it did not require intracellular calcium mobilization and did not alter the intracellular levels of cAMP. However, Cer 1-P induced strong and rapid phosphorylation of ERK1-2 in the macrophages, and inhibition of this pathway by PD098059 or UO126, which are selective inhibitors of mitogen-activated protein kinase kinase (MEK), blocked macrophage proliferation. In addition, Cer 1-P caused potent activation of PKB, as determined by its phosphorylation on Ser 473. PKB phosphorylation was blocked by the PI3-K inhibitor LY290042, suggesting that PKB is downstream of PI3-K in this system. Of importance, this PI3-Kinhibitor also blocked the mitogenic effect of Cer 1-P in the macrophages. We also found that treatment of the cells with Cer 1-P resulted in phosphorylation of glycogen synthase kinase-3 beta (GSK-3b), which is a kinase downstream of ERK1-2 or PKB. GSK-3b has been shown to stabilize the levels of proteins that are involved in the control of cell cycle, such as for instance cyclin D1 (CD1). Interestingly, Cer 1-P was also able to increase the expression of CD1 in the macropahges.
Taken together these results suggest that the PI3-K/PKB and MEK/ERK1-2pathways are implicated in the stimulation of macrophage proliferation by Cer 1-P.
This study was supported by grants BFU2006-13689/BFI from “Ministerio de Educación y Ciencia” (Madrid, Spain) and 9/UPV 00042.310-15852/2004 from “Universidad del País Vasco” (UPV/EHU) (Bilbao, Basque Country).

Sphinganine and sphinganine to sphingosine ratio as a biomarker of dietary fumonisins during chronic exposure in ducks.

O17
J. D. Bailly¹, D. Tardieu¹, A. Auvergne², R. Babilé², P. Guerre¹.
¹Department of mycotoxicology, National veterinary school of Toulouse, 23 chemin des capelles, 31076 Toulouse, France and ²National agronomy school of Toulouse, BP 107, 31326 Castanet Tolosan cedex, France.

Fumonisin B1 is structurally similar to sphinganine (Sa) and sphingosine (So) and inhibits ceramide synthase. It causes an elevation in Sa concentration, leading to an increase in Sa/So ratio in exposed animals. This increase often precedes fumonisins associated toxicity and that is why Sa/So ratio and free Sa have been proposed as biomarkers of fumonisin exposure in many species. The purpose of this study was to investigate the kinetics of Sa and So concentrations and Sa/So ratio in serum,liver and kidney over a 77 days exposure of ducks to different doses of FB1 to determine their use as biomarker of dietary fumonisins. The kinetics of 1P derivatives was also investigated in liver. One hundred and thirty mule ducks, 7 days of age, were randomly distributed into 5 groups receiving daily via oral route a dose of FB1 equivalent to ingestion of 0, 2, 8, 32 and 128 mg FB1/kg feed. On treatment days 7, 14, 21 and 28, 5 animals per group were bled and killed using carbon dioxide. The five remaining animals in each group were bled once a week during 7 further weeks. Neither mortality nor sign of mycotoxicosis were observed during the 77 days of treatment, no macroscopic nor microscopic lesions were observed on organs at the end of the treatment period. Determination of the kinetics of Sa/So ratio in serum revealed that Sa/So has increased dramatically from day 0 to day 7 (more than 3000% by using 128 mg FB1/kg feed). Then from day 7 to 28, a strong decrease occurred. The ratio continued to decrease slowly from day 28 to day 49 then stabilized throughout day 77. These variations were mainly due to modifications of Sa concentration in treated animals whereas only moderate effects were observed on So. Sa/So ratio in liver showed a very good correlation with that observed in serum. The kinetics was similar with a maximum reached at day 7 followed by a decrease until day 77. In the kidney, Sa/So analysis revealed that the ratio increased in all treated ducks to reach a maximum at day 7. Then after a slow decrease from day 7 to 14, the ratio stabilized until day 77. Liver sphinganine 1-phosphate was strongly increased in ducks fed 32 and 128 mg FB1/kg feed. Interestingly this increase is proportional to the amount of Sa within cells since Sa1P/Sa ratio remained nearly constant throughout the study, whatever the FB1 concentration in feed. In conclusion, although they can be considered as resistant to fumonisin toxicity, mule ducks are sensitive to the disruption of sphingolipid metabolism generated by these mycotoxins. However, this modification is also depending on the duration of exposure. Therefore, it seems that Sa/So ratio and Sa can be good biomarkers of exposure when ducks are exposed over short periods (1-2 weeks). If birds are exposed over 7 weeks, the determination of these parameters will probably fail to detect exposure. Although the mechanism of resistance of ducks to FB1 toxicity is still uncertain, it might be linked to the conversion of sphingoid bases into their 1P or other metabolite, and their elimination from the target tissues.

Purification and characterization of human intestinal neutral ceramidase.

O18
L. Ohlsson¹, C. Palmberg², R.-D. Duan¹, M. Olsson¹, T. Bergman², Å. Nilsson¹.
¹Gastroenterology and Nutrition Laboratory, Biomedical Centre B11, Lund University, S-22184 Lund, Sweden. ²Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.

Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus may have antitumour and anti-inflammatory effects in the gut. Although previous rodent studies1 including studies on neutral ceramidase (-/-) (ash2 -/-) mice2 indicate a role of neutral ceramidase in ceramide digestion, the human intestinal ceramidase has never been purified and characterized. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using14C-octanoyl-sphingosine as substrate. After four chromatographic steps, HiTrapQ, PD10, CHT hydroxyappatite and Superdex 200 HR chromatography, a homogenous protein band with molecular mass 116 kDa was obtained. MALDI mass spectrometry identified 16 peptide masses matching human mitochondrial ceramidase3. By RT-PCR and 5’RACE method, a predicted partial nucleotide sequence of neutral ceramidase was obtained from a human duodenum biopsy sample, which was homologous to that of known neutral/alkaline ceramidases. The enzyme has neutral pH optimum and catalyzes both hydrolysis and formation of ceramide in presence of bile salts. Distinct stimulation by any specific bile salt, was, however, not observed. The ceramidase activity is inhibited by Cu2+ and Zn2+ ions and by low concentrations of cholesterol. The enzyme is a glycoprotein but deglycosylation does not affect its activity. Our study indicates that neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut.
1Olsson et al., Am. J. Physiol. 2004 287:G929. 2Kono et al., J. Biol. Chem. 2006 281:7324. 3El Bawab et al., J. Biol. Chem. 2000 275:21508.

Effects of bile diversion in rats on intestinal sphingomyelinase and ceramidase.

O19
R. D. Duan¹, H.J. Verkade², Y. Cheng¹, R. Havinga², A. Nilsson¹.
¹Gastroenterology Laboratory, Biomedical Center, B11, Lund University, Lund, Sweden. ²Pediatric Gastroenterology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands.

Alkaline sphingomyelinase (Alk-SMase) and neutral ceramidase (N-Case) are localized in the intestinal microvillar membrane as ecto-enzymes and released into the gut lumen in an active form by bile salts and trypsin. The activities of these enzymes are also increased by bile salts at micellar concentrations. These enzymes have been shown to play important roles in sphingomyelin digestion in the small intestine and have implications in tumorigenesis in the colon. It is unclear to what extent that the intestinal presence of bile salts is critical for the intraluminal activities of these enzymes. In the present study, we compared the activities of Alk-SMase,N-CDase, and other types of SMases in control and permanently bile diverted rats. Bile deficiency rats were equipped with permanent catheters in bile duct and continuously bile drained. Animals were allowed to recover from surgery for 1 week and were subjected to overnight fasting before experiment. We found that in the intestinal tract of control rats, the activity of Alk-SMase was profoundly higher than those of acid and neutral SMases; the activity of neutral SMase in the lumen is very low, being less than 1% of that of Alk-SMase. Bile diversion reduced Alk-SMase activity by 85% in the small intestinal content, and by 68% in the faeces. Western blot showed that the reduced activity was caused by decreased level of enzyme protein in the intestinal lumen. In the mucosa of small intestine, the activities of acid and neutral SMase were only about 1-3 % of that of Alk-SMase. Bile diversion did not significantly affect Alk-SMase in the mucosa of small intestine. N-CDasedistributed in parallel with Alk-SMase in the intestinal tract. Bile diversion significantly reduced N-CDase activity both in the intestinal mucosa and content. The activity of alkaline phosphatase, another brush border enzyme, was not significantly changed in the intestinal mucosa by bile diversion. In conclusion, the intestinal presence of bile salts is important for maintaining high intraluminal levels of Alk-SMase and N-CDase, two key enzymes forstep-wise hydrolysis of sphingomyelin in the gut, and the digestion and absorption of sphingomyelin in cholestatic conditions could be impaired by the reduced hydrolytic capacities of the enzymes.

Influence of β-amyloid peptide, tumor necrosis factor-α and mexidole on the activity of neutral sphingomyelinase in rat brain.

O20
A.V. Alessenko, A.E. Bugrova.
Institute of Biochemical Physics of the Russian Academy of Sciences, 4 Kosygin str. Moscow 119991, Russia.

Alzheimer’s disease (AD) is characterized by progressive decline in cognition, memory and intellect. It has been hypothesized that amyloid-beta peptide (Aβ) and TNF-α may have prominent role in neurodegeneration. It is wellknown that neuronal death is developed according to apoptotic program. Most signaling pathway that trigger apoptosis remain unknown, but the sphingomyelin pathway has been recognized as a ubiquitous signaling system that links specific cell-surface receptors and environmental stresses to the nucleus. This pathway is initiated by the hydrolysis of sphingomyelin via the action of sphingomyelinases to generate ceramide. Ceramide then serves as a second messenger in this system, leading to apoptosis. Recently it was shown that cross-talk between oxidation system and sphingomyelin cycle exists in cells and could have important implication for the induction phase and evolution of AD. TNF-α activates receptors linked to multiple effector systems, including a sphingomyelin pathway and peroxide oxidation. Taking together the role of free radical reactions in pathogenesis of AD and participation of sphingomyelin pathway in apoptosis of neuronal cells, we have monitored induction of sphingomyelin cycle and activation of lipid peroxidation in rat brain sections after a single intracerebral injection ofAβ(25-35), TNF-α and their combination. For protection of brain against Aβ toxicity we injected mexidole- wellknown membraneprotector used for neurology for treatment ischemia and insult. We have determined the changes of neutral sphingomyelinase activity, sphingomyelin (SPM) and ceramide contents and level of lipid peroxide products (conjugated dienes and ketodienes) in the cerebral cortex, hippocampus and cerebellum of rats within 3 hrs and 7 days after Aβ (3 nmol per rat) and TNF- α (2 mkg per rat) intracerebral injection and mexidole after interperitonal injection in doses 100 and 200 mg per kg of rats. Maximal changes in the activity of sphingomyelinase, SPM, ceramide contents after single TNF-α and Aβadministration were found in the hippocampus, and were less expressed in the cerebral cortex and cerebellum. We found intensification of lipid peroxidation after 3 hrs of peptides injection to the rat brain, but activity of sphingomyelinase was not increased. Remarkable increases in sphingomyelinase activity and content of ceramide were found after 7 days of injection of Aβ. Increase in the level of peroxide products in hippocampus after 7 days of Aβ and TNF-α injection was still remarkable, but combinative injection of TNF-α and Aβ in the rat brain decreased activity of sphingomyelinase and level of ketodienes to normal level after 7 days. It was proposed that TNF-α may protect brain cells from injury induced by Aβ. The role of TNF-α in the pathogenesis of AD is unclear, because it has been shown to be involved in both neuroprotection and neurodegeneration depending on doses and age of animals. Small doses of this cytokine induced protective effect against Aβ neurotoxicity. In our experiments we used low dose of TNF-α. We suggest that using low doses of TNF-α in clinic for prevention of development of AD might be perspective. Mexidole decreased the level of lipid peroxides and activity of sphingomyelinase and content of ceramide in hippocampus. We suppose also that new drugs, protected brain cells from sphingomyelin digestion could be used for preventing or slowing the onset of AD.

Critical role for Sphingosine Kinase-1 in regulating survival of neuroblastoma cells exposed to amyloid-beta peptide.

O21
A. Gómez-Brouchet^1,2,3, D. Pchejetski^1,2,4, M.-L. Maddelein^2,4, V. García¹, M.- F. Altié¹, M.-B. Delisle^1,2,3, O. Cuvillier^1,2,4.
¹INSERM, U466, Toulouse, F-31000 France. ²Université Toulouse III Paul Sabatier, Toulouse, F-31000 France. ³CHU Toulouse, Service d’Anatomie et de Cytologie Pathologiques, Toulouse, F-31000 France. ⁴CNRS, Institut de Pharmacologie et de Biologie Structurale, UMR 5089, Toulouse, F-31000France.

The amyloid-beta (Aβ) peptide, the main constituent of amyloid plaques, is believed to play a causative role in the neurodegenerative process occuring in Alzheimer’s disease. Although Aβ-mediated neuronal cell death demonstrates biochemical characteristics of apoptosis, the molecular mechanism underlying Aβ toxicity remains largely undefined. Noteworthy, increased levels of ceramide have been found in the brain of Alzheimer’s disease patients, thereby implying that ceramide accumulation could contribute to Alzheimer’s disease pathogenesis. In addition Aβ toxicity was recently shown to be linked with ceramide generation both in cell culture models and in animal model. Compelling data have revealed the potential involvement of insulin-like growth factor I (IGF-I) in the Alzheimer’s disease pathophysiology. Low serum levels of IGF-I is correlated with premature brain amyloidosis and IGF-I has been found to regulate Aβ clearance from the brain. Furthermore, IGF-I prevents Aβ-induced neuronal cell death including inSH-SY5Y cells. The TGF- b1 peptide growth factor also protects neurons from a variety of insults including Aβ.
We examined the role of sphingosine kinase-1 (SphK1), a critical regulator of the ceramide/sphingosine 1-phosphate (S1P) biostat, in the regulation of death and survival of SH-SY5Y neuroblastoma cells in response to Aβ peptide(25-35). Upon incubation with Aβ, SH-SY5Y cells displayed a marked down- regulation of SphK1 activity coupled with an increase in the ceramide/S1P ratio followed by cell death. This mechanism was redox-sensitive as N- acetylcysteine totally abrogated the down-regulation of SphK1 activity and strongly inhibited Aβ-induced cell death. SphK1 overexpression impaired the cytoxicity of Ab while SphK1 silencing by RNA interference mimicked Aβ- induced cell death hence establishing a critical role for SphK. We further demonstrated that SphK1 could mediate the well-established cytoprotective action of IGF-I against Aβ toxicity through a PI3 kinase-dependent mechanism as wortmannin and LY294002 blocked SphK1 activation. A dominant-negativeform of SphK1 or its pharmacological inhibition not only abrogated IGF-I-triggered stimulation of SphK1 but also hampered IGF-I protective effect. Similarly to IGF-I, the neuroprotective action of TGF-b1 was also dependent on SphK1 activity as activation of SphK1 as well as cell survival were impeded by a dominant-negative form of SphK1. Collectively, these results provide the first illustration of SphK1 role as a critical regulator of death and survival ofAβ-treated cells.

Inhibition of sphingosine-1-phosphate-induced endothelial cell chemotaxis and PAF-synthesis by red grape and bilberry polyphenols.

O22
C. Barthomeuf^1,2, S. Lamy³, P. Chollet^1,4, R. Beliveau³.
¹INSERM-484, Université d’Auvergne, Centre hospitalier Jean Perrin, rue Montalembert, 63005 Clermont-Fd Cedex, France, ²Laboratoire de Pharmacognosie et Biotechnologies, Université d’Auvergne, Faculté de Pharmacie, Place H. Dunant, 63001 Clermont-Fd Cedex, France, ³Laboratoire de Médecine Moléculaire de l’Hôpital Sainte-Justine, Centre de Cancérologie Charles-Bruneau, Montréal, Québec, Canada and ⁴Unité de recherche clinique, Centre hospitalier Jean Perrin, Centre Anticancéreux, 63011 Clermont-Fd, France.

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid and a pro- inflammatory mediator, secreted mainly by platelets at clotting sites and, by several cell-types (including cancer cells) in response to various external stimuli. Five members of the endothelial differentiation gene (EDG) family have been identified as S1P receptors in a wide range of cell-lines. EDG- 1/S1P, a member of this receptor family, has recently been identified as an inducible transcript expressed during endothelial cell (EC) differentiation. By promoting the synthesis of endothelial platelet-activating factor (PAF) which stimulates EC migration, S1P enhances inflammation and pathological angiogenesis and, thereby, aggressive tumour proliferation and metastasis. We have recently reported that a red grape skin polyphenolic extract (SGE) preventsVEGF-induced endothelium neutrophil adherence1 and S1P-induced EC chemotaxis2 by decreasing the acute synthesis of PAF in S1P-stimulated ECs. These data provided the first demonstration that grape polyphenols may inhibit the S1P/endothelial differentiation gene-1 cascade. Inhibiting this cascade appears of great interest in cancer patients and in patients with cardiological disfunction. New experiments have been done to understand how SGE prevented S1P/EDG1 activation and,to compare the preventive effect of SGE and a commercially bioavailable highly purified bilberry extract (Antho50®) on S1P-induced HUVEC chemotaxis.Antho50® at 25 µM efficiently inhibited theserum-induced migration of androgen-resistant PC3 prostate cancer cells but, conversely to SGE, was unable to reverse the S1P/endothelial differentiation gene-1cascade activation in S1P-stimulated endothelial cells. Western blot, biochemical andMS-MS analysis revealed that SGE at 25 µM inhibited the immediate release of lysophophosphatidylcholine and of C16- and C18-alkyl PAF in HUVECs elicited by S1P. They correlated these effects with a lower activity and expression of group V secreted PLA2 (VsPLA2) in HUVECs’ membrane and a down-regulation of p38 MAPK and Akt in cells. In contrast to SGE, Antho50# at 25 µM was unable to inhibit the phosphorylation of p38 MAPK. These new data show that inhibitingV-sPLA2 activity and expression and subsequent p38 activation is critical for the prevention of S1P-induced signaling .
1 Barthomeuf et al., Bull. Cancer 2006 93:S123. 2Barthomeuf et al., Free Rad. Biol. Med. 2006 40:581.

Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion.

O23
L. Brizuela, M. Rábano, J. M. Macarulla, M. Trueba, A. Gómez-Muñoz.
Department of Biochemistry and Molecular Biology. Faculty of Science and Technology. University of the Basque Country. Bilbao. Spain.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid capable of regulating critical physiological and pathological functions. We have reported for the first time that S1P stimulates aldosterone secretion in cells of the zona glomerulosa of the adrenal gland. Regulation of aldosterone secretion is important because this hormone controls electrolyte and fluid balance and is implicated in cardiovascular homeostasis. S1P-stimulated aldosterone secretion was dependent upon the protein kinase C (PKC) isoforms α and δ and extracellular Ca2+ and it was inhibited by pertussis toxin (PTX). S1P activated phospholipase D (PLD) through a PTX-sensitive mechanism, also involving PKC α and δ and extracellular Ca2+. Primary alcohols, which attenuate the formation of phosphatidic acid (the product of PLD), and cell- permeable ceramides, which inhibit PLD activity, blocked S1P-stimulatedaldosterone secretion. Furthermore, propranolol and chlorpromazine, which are potent inhibitors of phosphatidate phosphohydrolase (PAP) (the enzyme that produces diacylglycerol from phosphatidate), also blocked aldosterone secretion. These data suggest that the PLD/PAP pathway plays a crucial role in the regulation of aldosterone secretion by S1P and that Gi protein-coupledreceptors, extracellular Ca2+, and the PKC isoforms α and δ are all important components in the cascade of events controlling this process.
This study was supported by grants 9/UPV 00042.310-15852/2004 from “Universidad del País Vasco” (UPV/EHU) (Bilbao, Basque Country), and BFU2006-13689/BFI from “Ministerio de Educación y Ciencia” (Madrid, Spain).

Internalisation of sphingosine-1-phosphate receptors.

O24
M. Jongsma, U. M. Florczyk, M. C. Hendriks-Balk, P. B. van Loenen, M. C. Michel, S. L. M. Peters, A. E. Alewijnse.
Department of Pharmacology and Pharmacotherapy, Academic Medical Center, Amsterdam, The Netherlands.

Internalisation is an important regulatory process that controls the signalling via G-protein coupled receptors. Upon ligand binding, receptors can be phosphorylated and translocated from the cell membrane to internal vessicles. From there, they are either recycled or broken down. Forsphingosine-1-phosphate (S1P) receptors, regulatory processes are of particular interest as there now is a drug in clinical trials which mechanism of action is suggested to involve downregulation of S1P1 receptors on T-cells. In this study we aimed to compare internalisation of different S1P receptor subtypes, which requires a sensitive and quantitative assay. While several methods have been used to study receptor internalisation, none of them allows a precise quantitative description. We have therefore set up a new method to quantitatively measure internalisation with N-terminal HisG- tagged receptors stably transfected into CHO-FlpIn cells. Agonist exposure caused internalisation of S1P1, S1P2 and S1P3 receptors, but that of the S1P3receptor differed from that of the other subtypes in several ways: It required much lower S1P- concentrations despite S1P having comparable affinity and potency at all subtypes. While the S1P concentrations required to increase intracellular calcium and/or affect cAMP accumulation were similar to those required for internalisation at the S1P3 receptor, the other subtypes required a much higher S1P concentration to induce internalisation than to activate signalling. Moreover, S1P3 receptor internalisation was much faster than that of S1P1 and S1P2 receptors, requiring 5 min vs. up to 30 min. This may be explained by a recent finding that internalisation of the S1P3 receptor is independent of phosphorylation in contrast to that of the S1P1 receptor. Preliminary results suggest that also resensitisation differs among these three S1P receptor subtypes. In conclusion, our study shows that S1P1, S1P2 and S1P3receptors differ markedly with regard to agonist-induced internalisation. We hope to elucidate the mechanisms responsible for these differences in future studies.

Sphingosine-1-phosphate induces apoptosis in lyase deficient primary cultured murine cerebellar neurons.

O25
N. Hagen¹, R. Broere¹, P. P. Van Veldhoven², G. van Echten-Deckert¹.
¹Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universitaet Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany. ²Katholieke Universiteit Leuven, Campus Gasthuisberg, Afdeling Farmakologie, Herestraat Box 601, B-3000 Leuven, Belgium.

Sphingosine-1-phosphate (S1P) was shown to regulate a wide range of physiological processes including proliferation, differentiation, motility, cytoskeleton rearrangements and calcium homeostasis1. Using cis-4-methylsphingosine, a synthetic sphingosine analogue pro-drug that is phosphorylated intracellulary to a metabolically stable S1P analogue, we have, however, demonstrated that in terminally differentiated post-mitoticneurons S1P has an apoptotic rather than an anti-apoptotic effect2. Accordingly, primary cultured neurons from 6-days-old lyase deficient mice, which are unable to cleave phosphorylated sphingosine, underwent apoptosis in response to exogenously added S1P. As in case of the lyase resistant phosphorylated cis-4-methylsphingosine, an abortive reactivation of the cell cycle on the one hand and activation of caspases on the other hand appear to be essential for S1P-induced apoptosis in lyase deficient neurons. Note that lyase deficient mice exhibit a complex phenotype including pronounced growth impairment and shortened life span that generally does not exceed the nursing period3.
The (patho)physiological significance of our observations is not yet clear but neuronal death could be indicative for an involvement of S1P in neurodegenerative disorders.
1Spiegel and Milstien, Nat. Rev. Mol. Cell. Biol. 2003 4:397. 2Naetzker et al., Genes Cells 2006 11:269. 3Van Veldhoven, Chem. Phys. Lipids 2005 136:164.

Sphingosine kinase-1 is a downstream regulator of imatinib-induced apotosis in LAMA84 chronic myeloid leukemia cells.

O26
E. Bonhoure^1,2, A. Lauret¹, B. Malavaud^2,3, T. Kohama⁴, J. Melo⁵, O. Cuvillier^1,2.
¹INSERM, U466, Toulouse, F-31000 France, ²CNRS, Institut de Pharmacologie et de Biologie Structurale, UMR5089, Toulouse, F-31000 France, ³Université Toulouse III Paul Sabatier, Toulouse, F-31000 France, ⁴Sankyo Co. Ltf., Core Technology Research Laboratories, Tokyo, 140-8710, Japan and ⁵Department of Haematology, Falculty of Medicine, Imperial College London, Hammersmith Hospital, London, United Kingdom.

Chronic myeloid leukaemia (CML) is a myeloproliferative disease characterized by the expression of the oncogenic fusion protein tyrosine kinase BCR-ABL that is essential for leukemogenesis and which occurs in 95% of CML. Inhibition of this tyrosine kinase activity appears to be the most effective way to abolish the activity of this oncoprotein. Imatinib mesylate (or STI571), a specific inhibitor of BCR-ABL, exhibits potent antileukemic effects in vitro and in vivo, and is now the first-line treatment for CML. Despite imatinib is highly effective in the treatment of CML, the emergence of resistance has now become a significant issue. A better understanding of the signaling pathways initiated by imatinib in CML cells is a prerequisite step in order to develop new therapeutic strategies to treat patients who do not respond anymore to imatinib.
In our study, we examined the involvement of the oncogenic sphingosinekinase-1, a critical regulator of the ceramide/sphingosine 1-phosphatesphingolipid balance, in the imatinib resistance of LAMA-84 cells. Contrary to sensitive LAMA-84s cells, imatinib failed to induce apoptosis in chemoresistant LAMA-84r cells overexpressing Bcr-Abl. The chemosensitivity of LAMA-84s to imatinib was associated with a decrease in sphingosinekinase-1 activity and S1P level and an increase in ceramide level. In contrast, chemoresistant LAMA-84r had sustained sphingosine kinase-1 activity and did not produce ceramide during treatment with imatinib. Overexpression of sphingosine kinase-1 in LAMA-84s cells resulted in marked inhibition ofimatinib-induced apoptosis.
It is well established that the Bcr-Abl signaling involves the Ras/Raf/MEK/ERK pathway. Inhibition of Ras and MEK has been shown to circumvent imatinib- induced resistance. Herein, we show that Ras farnesyl-transferase inhibitor (FTI) and MEK inhibitor (UO126) triggered sphingosine kinase-1 inhibition during apoptosis of LAMA-84r cells. Enforced expression of sphingosine kinase- 1 prevented cell death induced by FTI and UO126. To further establish the critical role for sphingosine kinase-1 in regulating CML apoptosis, we next studied the effect of cytosine β-D-arabinofuranoside (Ara-C), a known CML chemotherapy that has recently been shown to overcome imatinib-resistancein the LAMA84-r cell model. Interestingly, Ara-C induced marked inhibition of sphingosine kinase-1 activity during apoptosis, and sphingosine kinase-1overexpression counteracted its effect.
We next established the proof-of-concept that sphingosine kinase-1 inhibition is an essential event to mediate apoptosis of CML cells by showing that sphingosine kinase-1 inhibition (pharmacological inhibitor, RNAi strategy) could kill CML cells regardless of their imatinib-resistance status. Overall, this work demonstrates that sphingosine kinase-1 could be a good target for efficiently overcoming the imatinib-resistance problem in CML.

The pleiotropic effects of S1P are mitigated by an anti-S1P mAb in multiple murine models of cancer, age-related macular degeneration and heart failure.

O27
R. A. Sabbadini.
San Diego State University and Lpath, Inc., San Diego, CA, USA.

It has been recognized that alterations in lipid metabolism can lead to cancer, cardiovascular disease, diabetes, neurodegenerative disorders, immune function, pain, mental disorders and inflammation. However, only as a consequence of our recent appreciation that bioactive lipids are bona fide signaling molecules have key members of the functional lipidome been viewed as targets for rational drug design in mitigating lipid-associateddisorders. Recent research has demonstrated that the bioactive signaling molecule, sphingosine-1-phosphate (S1P) plays a pivotal role in cancer progression. We hypothesize that S1P may also be associated with maladaptive wound healing and angiogenesis in various ocular and cardiovascular disorders as well. We have developed a bio-specificmonoclonal anti-S1P antibody (anti-S1P mAb) that could be used as a therapeutic molecular sponge to selectively neutralize S1P. The therapeutic potential of the anti-S1P mAb was analyzed in several murine models of cancer as well as a model of Age-related Macular Degeneration (AMD) and in awell-established murine model of heart failure. For cancer, theanti-tumorigenic effects of the anti-S1P mAb were evaluated in multiple cell lines representing a spectrum of histological cancer subtypes. The ability of S1P to stimulate proliferation, promotes cell invasion and protects tumor cell from apoptosis induced by chemotherapeutic drugs was neutralized by theanti-S1P mAb. We also demonstrate the ability of our antibody to inhibit angiogenesis. The anti-S1P mAb blocked both the migration of endothelial cells and their ability to form de novo capillary-like structures that resemble blood vessels using in vitro Matrigel models. In vivo, we investigated the ability of the anti-S1P mAb to reduce tumor volume and inhibit angiogenesis in multiple murine models. The anti-angiogenic effects of the antibody were also validated in a murine model of the ocular disorder, AMD, induced by laser burn or Bruch’s membrane. The resulting choroid neovascular lesions were almost completely prevented by intravitreal injection of the antibody. Moreover, the profibrotic effects of S1P were mitigated by this treatment such that substantial reductions in scarring, inflammation and other aspects of maladaptive wound healing were observed. The anti-scarring effect of theanti-S1P mAb was also studied in a murine model of post-MI heart failure, a condition associated with maladaptive fibrosis and scarring that results in cardiac dysfunction and death. The antibody dramatically reduced scarring and improved cardiac function and animal survivability. Taken together, the findings from multiple murine disease models suggests that the anti-S1PmAbis not only a useful research tool to investigate S1P’s role in tumorigenesis, angiogenesis and maladaptive wound healing, but it may represent a novel and innovative approach to the treatment of disease. The anti-S1P mAb may be the first example of targeting an extracellular bioactive lipid for use as a molecular sponge to prevent key ligand-receptor interactions. This leads one to argue that the antibody may be a first-in-class drug of an emerging discipline that may be termed lipidomic- based therapeutics.

Critical role of acidic sphingomyelinase in murine hepatic ischemia reperfusion injury.

O28
L. Llacuna, M. Marí, R. Paris, C. García-Ruiz, J. C. Fernandez-Checa, A. Morales.
Liver Unit, Hospital Clinic, IDIBAPS, IIBB-CSIC, Barcelona, Spain.

Hepatic ischemia reperfusion (I/R) damage can occur in diverse settings including liver transplantation, trauma, hemorrhagic shock or liver surgery, and represents a serious clinical complication that may compromise liver function because of extensive hepatocellular loss. However, the molecular mechanisms of hepatic ischemia reperfusion (I/R) damage are not completely known. Thus, we investigated the role of ceramide in a murine model of warm hepatic I/R injury, as this sphingolipid induces cell death and participates in TNF signaling. Determination of hepatic ceramide homeostasis and its metabolic regulation during I/R. Inhibition or suppression of ceramide- regulating enzymes and consequences in hepatic I/R damage. Hepatic ceramide levels transiently increased after the reperfusion phase of the ischemic liver in mice, due to an early activation of acidic sphingomyelinase (ASMase) followed by acid ceramidase stimulation. In vivo administration of an ASMase inhibitor, imipramine, or ASMase knockdown by siRNA, which decreased ceramide generation during I/R, significantly attenuated serum ALT levels, hepatocellular necrosis, cytochrome c release and caspase-3 activation, by preventing JNK activation and Bim translocation to mitochondria. In contrast, blockade of ceramide catabolism by N- oleyolethanolamine (NOE), a ceramidase inhibitor, enhanced ceramide levels and potentiated I/R injury compared to vehicle-treated mice. Pentoxifylline treatment prevented both TNF up-regulation and ASMase activation and protected the liver against I/R injury. Moreover, 80-90% of rats treated with imipramine survived 7 days after total liver ischemia compared to 30-40%survival of vehicle-treated animals, while 100% NOE-treated rats died within 2 days of total liver ischemia. Ceramide generated from ASMase plays a key role in I/R-induced liver damage and its modulation may be of therapeutic relevance.

Searching for biomarkers of Gaucher disease using proteomic tools.

O29
L. Quintana¹, A. Monasterio², L. Simón², A. Martínez², P. Giraldo¹, M. Pocoví¹.
¹Instituto Aragonés de Ciencias de la Salud, Edificio CEA, Avda. Gómez Laguna 25, 50009 Zaragoza. ²Proteomika SL, Parque Tecnológico de Vizcaya, Edificio 801B, 48160 Derio.

Introduction. Gaucher disease (GD) is a lysosomal glycolipid storage disease caused by mutations in the acid β-glucosidase gene that shows autosomal recessive inheritance. This defect causes a deficiency in the glucocerebrosidase enzyme that leads to the accumulation of glucocerebroside in the lysosomes of macrophages. Many biomarkers show altered accumulation in the plasma of GD patients and can be used for disease diagnosis and follow-up. Chitotriosidase protein (ChT) is the most important biomarker described in GD. However, its clinical application is restricted because a common genetic defect that results in the absence of detectable ChT in the plasma of 6% of Caucasian population. Thus, new surrogate biomarkers are necessary for effective diagnosis and to decide therapeutic intervention, such as the recently described chemokine CCL18/PARC. Proteomics has recently emerged as a new technology for global analysis of protein profiles and biomarker identification in biological fluids. This technology can play a critical role in the elucidation of GD pathophysiology and for monitoring patient response to therapies.
Materials and Methods. In this work, a population of 39 individuals was studied for differentially expressed plasma proteins grouped as follows: 16 GD patients and 23 healthy controls. For patient sample collection, plasma was retrieved immediately following diagnosis and prior to treatment. Plasma samples were analysed using proteomic tools: proteins were resolved by two dimensional gel electrophoresis (2-DE) and silver stained. Image analysis was performed using Progenesis PG220 software (Nonlinear Dynamics). This analysis includes detection, quantization and normalization of each protein spot in every 2-DE image. Significant differences in protein expression levels between controls and GD patients were determined by t Student test with a set significance value of p < 0.05.
Results. Following this methodology a set of 30 differentially-expressedplasma proteins has been identified by mass spectrometry. This set includes proteins that are involved in regulation of immune system and inflammation, metabolism of lipoproteins and coagulation cascade. Potential plasma biomarkers of GD are being still validated using specific antibodies bywestern-blot.

RNAi-mediated inhibition of the glucosylceramide synthase (GCS) gene: a preliminary study towards a therapeutic strategy for Gaucher disease and other glycosphingolipid storage diseases

O30
A. Diaz-Font¹, A. Chabás², D. Grinberg¹, L. Vilageliu¹.
¹Departament de Genètica, Facultat de Biologia, Universitat de Barcelona and ²Institut de Bioquímica Clínica, Corporació Sanitària Clínic. Barcelona, Spain.

In the last few years, small interference RNAs (siRNAs) have been used in a number of different experimental settings to silence gene expression. In some of them, chemically synthesized or in vitro transcribed siRNAs have been transfected into cells. In others, siRNAs have been expressed endogenously from siRNA expression vectors. Enzyme replacement and substrate deprivation therapies are the current approaches to treat Gaucher disease. Although good results have been reported there are several limitations and side effects that make interesting to search for new alternatives. We present here a new approach based on the inhibition of theGCS gene using siRNAs as a possible future therapeutic strategy for Gaucher disease. We have designed four siRNAs for the human GCS gene and transfected them into HeLa cells. With two of them, a clear reduction of GCSRNA levels and enzyme activity was obtained. Consistently, a reduction in glucosylceramide synthesis was observed. Similar results were obtained when plasmids expressing shRNAs (targeting the same sequences) were transfected into the cells. The inhibition of the mouse homolog Ugcg gene was also achieved, using a siRNA that targeted both human and mouse sequences. In summary, we have shown that siRNAs are a good tool to silence GCS gene expression and, thus, to reduce glucosylceramide formation. This success at the cellular level, including human and mouse cells, should be followed by experiments in animal models to develop a new therapeutic strategy for Gaucher disease.

The Molecular Characterization of Gaucher Disease in Spain.

O31
P. Alfonso^1,3 M. Pocovi^1,3, P. Giraldo^2,3.
¹Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain. ²Miguel Servet University Hospital, Zaragoza, Spain. 3Institute Aragones of Health Sciences (I+CS), Zaragoza, Spain.

Mutations in the β-glucocerebrosidase (GBA) gene cause Gaucher disease (GD), an autosomal recesive inherited disorder associated to organomegaly, thrombocytopenia, anaemia, bone disease and in the most severe cases neurological involvement. The aim of this study was to characterize the GBA mutations in a group of 193 apparently non-related from the Spanish GD Registry. We have screened all patients for the presence of N370S and L444P previously described mutations. To identify other unknown mutations the promoter region and the 11 exons with their flanking intron sequences of the GBA gene were screened by DNA sequencing. The allelic frequencies of Spanish are reported and the mutation profile is analyzed. This approach led to the identification of 98.7% of mutant GBA alleles. We have found 55 different GBA mutations and 65 genotypes causing GD in Spain: 49 mutations previously described, and 9 novel mutations: 4 missense R395C, R463H, W312R, V398I; one nonsense R359X, 3 frame-shift c.708delC, c.1214- 1215delGC, c.1439-1445del7, and one in-frame c.42-65 del 24. The most prevalent mutations were N370S and L444 P accounting for the 68.7 % of the mutant alleles. Among the type 1 GD patients, 93.8 % carried the mutation N370S. The wide phenotypic differences observed within the genotypic groups as well as between siblings implicate a significant contribution of other modifying genetic and/or non-genetic factors. All of these findings indicate that there is significant genotypic heterogeneity among Spanish GD patients.
*on behalf of the Spanish Registry of Gaucher’s disease.

Effective Gene Therapy in a Mouse Model of Sandhoff Disease.

O32
B. Cachón-Gonzaléz¹, S. Z. Wang¹, R. Ziegler², S. H. Cheng², T. M. Cox.1
¹Department of Medicine, University of Cambridge, Cambridge, CB2 2QQ, UK and ²Genzyme Corporation, Framingham, MA, 01701-9322, USA.

The inborn neurodegenerative Tay-Sachs and Sandhoff diseases are caused by mutations in the α- and β-subunits of β-hexosaminidase A (αβ) [HexA] and B (ββ) [HexB] isozymes, respectively. In the absence of active enzyme, GM2 ganglioside and related glycosphingolipids accumulate prominently in the lysosomes of neurones. This storage is associated with inflammation, cell loss and neurological dysfunction.
The Sandhoff mouse, homozygous for a disrupted β-hexosaminidase β subunit allele (hex β -/-), serves as a model of human GM2 gangliosidosis: although normal at birth, it develops a neurodegenerative disease requiring euthanasia by the age of 16 weeks.
We have used AAV-derived vectors to direct expression of human hex β (hhexβ) and hex α (hhexα) in the brain of Sandhoff mice in an attempt to improve outcome by gene therapy and thereby further to investigate the pathogenesis of this disease.
One-month old Sandhoff mice were injected with a mixture of AAV2/2 expressing hhexβ (AAVhhexβ) and hhexα (AAVhhexα) or AAVhhexβ and AAVhhexα singly, in the presence of 6% mannitol. Single striatal injections, as well as four-site injections (two in the striatum and two in the cerebellum), were performed. All animals treated with AAVhhexα and β or AAVhhexβ alone, and culled at different post-injection times, showed widespread therapeutic gene expression ipsilaterally and contralaterally. Pathological glycolipid storage was cleared with reduced microglial expansion/activation and fewer apoptotic cells. The onset of symptoms in six untreated Sandhoff mice was 9613 (sd) days; they reached their humane end-point at 1204 days. Mice receiving control injections of AAVhhexα alone had a similar evolution with symptom onset and end points at 101 8 and 120 5 days, respectively. In contrast, six mice injected with a mixture of AAVhhexα and AAVhhexβ at a single site had delayed symptoms and reached the humane end point at 19917 days. More than 40% of animals receiving four-site injections of AAVhhexβ alone survived beyond one year, (p<0.0001).
In this acute model of Sandhoff disease, our gene therapy strategy greatly increased survival and improved neuronal function with concomitant maintenance of motor coordination, balance and body weight – onset of ataxia and tremor was delayed. The intervention also decreased neuro- inflammatory and apoptotic responses, as well as glycosphingolipid storage.Co-injection of AAVhhexα with AAVhhexβ was not required for this salutary outcome.

Cyclodextrin-Mediated Chiral Separation of Sphingolipids and High Resolution Detection by Capillary Electrophoresis.

P1
B. M. Barth^1,2,3, D. L. Kirschner^2,3, T. B. Kuhn^1,2,3, T. K. Green^2,3.
¹Alaska Basic Neuroscience Program, ²Institute of Arctic Biology and ³Department of Chemistry and Biochemistry, University of Alaska-Fairbanks,Fairbanks, Alaska 99775 USA.

Sphingolipids are a unique and major class of lipids important not only to membrane structure and dynamics, but also to cellular function and signaling. The bioactive properties of sphingolipids are as diverse as the lipids themselves and range from mediation of survival and development to apoptosis. Numerous analytical techniques have been developed to study sphingolipids, yet the ability to analyze chirality of sphingolipids in biological samples remains rather impractical due to the large amount of sample often needed. In this study, we sought to develop a new method to detect and analyze sphingolipids that would be applicable to small biological samples.
Capillary Electrophoresis (CE) is a powerful analytical tool for high resolution analysis of biological species including amino acids, peptides, and viruses. Micellular Electrokinetic Chromatography (MEKC) is a commonly employed CE technique often used for separation of hydrophobic molecules with charged or neutral functional groups. Cyclodextrins (CDs) are water soluble macrocyclic sugars commonly used as buffer additives in MEKC to improve separation efficiency and allow for chiral resolution. The CDs act by forming chiral host- guest inclusion complexes with the hydrophobic guest molecules. To date, we are unaware of any CE methods developed for analysis of sphingolipids.
In this study we derivatized a mixture of D and L threo-dihydrosphingosinewith naphthalene-2,3-dicarboxaldehyde (NDA) and sodium cyanide to yield the highly fluorescent cyanobenz[f]isoindole derivatives. The analytes were separated and detected using CE with laser induced fluorescence (LIF) detection. Various buffer parameters affecting separation were investigated including a combination of 30 mM SDS and 20 mM α-CD, which achieved baseline resolution of dihydrosphingosine. The method was applied to the analysis of other sphingolipids and amino acids as well.
Lastly, biological samples obtained from developing chicken embryos were investigated utilizing this unique CE method. Altogether, our research is an initial step towards the development of a unique method that allows for accurate and quantifiable low level detection of various chiral sphingolipids in biological samples.

Changes in glycosphingolipid content and overexpression of glucosyl- ceramide synthase are associated with acquired resistance to unrelated drugs in T98G human glioma cells.

P2
R. Bassi, P. Giussani, V. Anelli, P. Viani, L. Riboni.
Department of Medical Chemistry, Biochemistry and Biotechnology, LITA- Segrate, Milan, Italy .

Different studies support that glycosphingolipids (GSLs) act as important players in tumor biology, and emerging evidence suggests their involvement in drug resistance too. Glucosylceramide synthase (GCS) is a pivotal enzyme in sphingolipid metabolism which converts ceramide to glucosylceramide (GlcCer). In drug-resistant cancer cells and in tumor specimens from patients with scarce response to chemotherapy, the activity of GCS and the levels of GlcCer were found elevated. Moreover, inhibitors of GCS have been shown to increase the cellular levels of ceramide, a key player in the regulation of apoptosis, and to reverse drug resistance. Notwithstanding, little is known on the involvement of GCS in malignant gliomas. These are the most frequent and deadly human primary brain tumors, their intrinsic or acquired resistance limiting therapy effectiveness. In this study, we investigated the role of GCS in the resistance of T98G human glioma cells to paclitaxel and temozolomide, two unrelated drugs with clinical activity against gliomas. By selection with gradually increasing drug concentrations, we generated a paclitaxel (Tax-R)and a temozolomide (Tmz-R) resistant cell line. The analysis of resistance markers by immunoblotting showed that MDR1 (P-gp) expression was exclusive of Tax-R cells, whereas alkylguanine methyltransferase was present in all cell lines, and overexpressed exclusively in Tmz-R cells. After labeling at equilibrium with 3H-sphingosine or 3H-serine, Tax-R and Tmz-R were both characterized, with respect to sensitive T98G, by increased levels of both GlcCer and gangliosides. On the contrary, the levels of lactosyl-ceramide, the major neutral glycosphingolipid of T98G cells, were significantly lower inTmz-R, but not in Tax-R cells. In addition, the ceramide levels were found lower in resistant vs. sensitive cells. In both Tax-R and Tmz-R cells the in vitro activity of GCS was significantly higher than in sensitive cells. Moreover, a semi-quantitative RT-PCR analysis showed that a higher expression of the mRNA for the GCS gene was evident in both resistant cell lines. Finally, cytotoxicity assays revealed differences between the cell lines with respect to their sensitivity toward GCS inhibitors, cell survival in resistant cells being significantly lower than in sensitive ones. Noteworthy, exposure of Tmz-R cells to the GCS inhibitor PDMP restored cell sensitivity to temozolomide. Altogether our data demonstrate that overexpression of GCS and alterations of glycosphingolipid level, with increased GlcCer and ganglioside content, are associated to T98G resistance to unrelated cytotoxic drugs. These variations occur independently of MDR1 expression, and are associated to the attenuation of ceramide levels. This suggests that the increase of specific glycosphingolipids might offer glioma cells a survival advantage, resulting in resistance to chemotherapeutic drugs.
Grant support: MURST PRIN 2004 to LR.

Fluorimetric determination of sphingosine-1-phosphate lyase activity.

P3
C. Bedia, J. Casas, G. Fabrias.
Research Unit on BioActive Molecules, Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18, 08034-Barcelona, Spain.

Intracellular sphingosine 1-phosphate (S1P) levels are regulated by its synthesis from sphingosine by sphingosine kinases and its catabolism by lipid phosphatases and by sphingosine 1-phosphate lyase (SPL). The latter is a pyridoxal 5-phosphate-dependent enzyme residing in the endoplasmic reticulum and is responsible for the irreversible retroaldolic cleavage of S1P to ethanolamine phosphate and hexadecenal. The notion that inhibition of sphingosine 1-phosphate lyase may account for some effects of FTY720 on immune function1 supports that SPL may be a potential target for immunomodulatory therapy and that the search for potent and selective inhibitors of SPL may offer novel immunosupressant drugs. The discovery of SPL inhibitors requires the availability of a reliable enzyme assay. The measurement of the SPL activity has been carried out for years using radiolabeled dihydrosphingosine as substrate in an poorly reproducible assay. In this work we describe the development of a feasible and consistent procedure to determine SPL activity. This method uses a fluorogenicsphinganine-1-phosphate analog as substrate, which, after the carbon-carbonbond cleavage produced by SPL, releases an aldehyde, which in turn suffers a spontaneous β-elimination to liberate the fluorescent molecule umbelliferone. The kinetic parameters of the SPL-mediated cleavage of this substrate and the implementation of the method for the high throughput screening of libraries of compounds both in vitro and living mammalian cells are reported.
1Bandhuvula et al., J. Biol. Chem. 2005 280:33697.

Cholesterol and Sphingomyelin relationship in the cancer.

P4
I. Bernardini, L. Pugliese, L. Cecchetti, E. Bartoccini, M. P. Viola Magni, E. Albi.
Department of Clinical and Experimental Medicine, Physiopathology, University of Perugia. Italy.

In the last thirty years it has been described a strong hypocholesterolemy in patients affected with tumour not due to synthesis decrease1. Numerous studies have shown that exists a strong interaction between unesterified cholesterol (CHO) and sphingomyelin (SM) which arises from the Van der Waals interaction. Since SM was important in the physiological and pathological proliferation, the aim of the present work was to study the possible hyposphingomyelinemia associated to hypocholesterolemia in the patients with cancer. The blood of not drug treated 25 patients with monoclonal gammopathy were analyzed for their proteins and lipids content. The results demonstrated that the group with high level of gamma proteins presented a strong decrease of CHO and SM in blood. To investigate about the possible mechanism, we have evaluated the content of CHO, CHO esters and SM in the culture medium culture of SUP-T1 lymphoma cells at 0 and 48 hours of culture. The results showed that there was a strong reduction of CHO and SM without a variation of CHO esters accompanied by an increase of these lipids inside the cells. Enriching the culture medium by 800nM CHO, the cell content of CHO-SM increased together with an increase of 60% 3H-tymidineincorporation. The data suggest that the tumour cells need to incorporate these lipids to growth and that it could to explain the hypocholesterolemia and hyposphingomyelinemia in patients with cancer.
1Pugliese L. et al Albi E. Int.J. Immunopath. Pharm. 2006, in press.

Sphingosine 1-phosphate stimulates aldosterone secretion through activation of the PI3-K/PKB and MEK/ERK 1/2 pathways.

P5
L. Brizuela, M. Rábano, J. M. Macarulla, M. Trueba, A. Gómez-Muñoz.
Department of Biochemistry and Molecular Biology. Faculty of Science and Technology. University of the Basque Country.

The Phosphoinositide 3-kinase/protein kinase B (PI3-kinase/PKB) pathway and extracellular signal-regulated kinases 1/2 (ERK1/2) play essential roles in the regulation of cell growth and survival, chemotaxis, cell motility, and secretion. These pathways can be activated by a variety of agonists, including bioactive sphingolipids. In particular, sphingosine 1-phosphate (Sph 1-P) has been implicated in the regulation of cell proliferation and survival, cell differentiation, tumour cell invasion, smooth muscle contraction and angiogenesis. Of interest, our group has preliminary evidence suggesting that Sph 1-P might be involved in steroidogenesis. Although Sph 1-P can act intracellularly as a second messenger, many of its effects are elicited through interaction with specific Gi protein- coupled receptors (S1P1-5) that are ubiquitously expressed in cells. In the present work, we show that Sph 1-Pstimulates aldosterone secretion in glomerulosa cells of bovine adrenal glands, and that the PI3-kinase/PKB and ERK 1/2 are essential pathways implicated in the regulation of this process.
This study was supported by grants 9/UPV 00042.310-15852/2004 from “Universidad del País Vasco” (UPV/EHU) (Bilbao, Basque Country), and BFU2006-13689/BFI from “Ministerio de Educación y Ciencia” (Madrid, Spain).

In vitro, phytosphingosine enhances the cytotoxicity of drugs used forhormone-refractory prostate cancer treatment.

P6
A. Cabrespine¹, E. Debiton², J.-O. Bay¹, P. Chollet¹, C. Barthomeuf².
¹Centre Jean Perrin, Unité de recherche clinique Clermont-Ferrand, France; UMR484 INSERM, Clermont-Ferrand (France) and ²Université d’Auvergne, Laboratoire de Pharmacognosie et Biotechnologies, UMR484 INSERM,Clermont-Ferrand (France).

Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer-related death of men in industrialized countries. Chemotherapy is the treatment of choice for patients with hormone- refractory prostate cancer (HRPC). Mitoxanthrone, carboplatin and paclitaxel have demonstrated some clinical value. However, in monotherapy, they have only limited efficacy. Combined chemotherapy appears a valuable approach to enhance their clinical value. In vitro drug combination studies conducted in conditions that mimic what happened in vivo could be helpful in the design of protocols that would have greater clinical efficacy. We previously reported that combination of paclitaxel and 13-cis-retinoic acid and of chelerythrine, mitoxanthrone and paclitaxel, may have interest for the treatment of HRPC.1 Phytosphingosine (PS) induces apoptotic cell death via caspase 8 activation and Bax translocation in human T-cell lymphoma (Jurkat) and in NCI-H460 non-small cell lung cancer cells.2 In combination with ionizing radiation, PS also enhances apoptotic cell death inradiation-resistant cancer cells through ROS-dependent and –independent AIF release.3 As these mechanisms may be helpful for cancer treatment, we have developed a mutant fungic strain overexpressing PS and a purification process leading to obtain high amounts of pure PS (Barthomeuf, personal communication). The aim of this study was to evaluate the value of PS in combination with mitoxanthrone, carboplatin, 13- cis-retinoic acid or paclitaxel vs PS alone and vs each drug alone or in combination. Tests were conducted on PC3 androgen-independent prostate cancer cells cultured insteroid-free conditions. Synergism, additivity and antagonism were determined by the combination index and calculated for each combination by the median-effect method. Data confirm that in combination, this sphingoid base may have clinical value for the treatment of HRPC.
1Cabrespine et al., Anticancer drugs 2005 16:417. 2Park et al., Clin. Cancer Res. 2003 9:878.3Park et al., Blood 2005 105:1724.

Sphingolipid analogs: Searching new drugs against COPD.

P7
D. Canals,¹ G. Villorbina,¹ S. Grijalvo^1,2, A. Delgado^1,2, A. Llebaria,¹ G. Fabriàs,¹ J. Casas¹.
¹Research Unit on BioActive Molecules, Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18, 08034-Barcelona, Spain and ²University of Barcelona, School of Pharmacy, Unit of Pharmaceutical Chemistry (CSIC Associated Unit), Juan XXIII, s/n, 08028, Barcelona, Spain.

Chronic obstructive pulmonary disease (COPD) is characterised by chronic inflammation of the airways and progressive destruction of lung parenchyma, a process that in most cases is initiated by cigarette smoking. Among the several mechanisms postulated to be involved in the pathogenesis of COPD, disruption of the balance between apoptosis and replenishment of structural cells in the lung has been recently reviewed.1 There is an increase in apoptotic alveolar epithelial and endothelial cells in the lungs of COPD patients. Moreover, Petrache et al reported increased lung ceramide levels in emphysema patients, suggesting that ceramide upregulation might be an important pathogenetic element in the development of emphysema.2
The putative apoptotic activity of D-erythro-dihydroceramides (DHC’s) is a matter of controversy. Thus, it has been published that exogenously added short chain DHC’s do not exhibit apoptotic activity in different cell lines,3,4while L-threo-N-acetyl-sphinganine causes cell death in HL-60 cells.3Additionally, accumulation of natural DHC’s, but not ceramides, along with cell death occurs in human leukaemia cells treated with gamma-tocopherol,as well as in prostate cancer cells.5 Finally, a recent report describes that both long and short chain erythro-DHC’s do not simply lack apoptogenic activity, but counteract the apoptotic effect of ceramide by inhibiting Cer channel formation in mitochondria in early apoptosis.6
In this context, the synthesis of a small combinatorial DHC library obtained by systematic variation of both the sphingoid and aliphatic and acyl chains is reported in this work. In addition, preliminary data on the activity of the synthesized compounds in A549 cells, a human alveolar epithelial cell line used in COPD studies, are also presented.
1Demedts et al., Respir. Res. 2006 30:7. 2Petrache et al., Nat. Med. 2005 11:471. 3Bielawska et al., J. Biol. Chem. 1993 268:26226. 4Ogretmen et al., J. Biol. Chem. 2001 276:32506; 5Jiang et al., Proc. Natl. Acad. Sci. USA 2004 101:17825. 6Stiban et al., Apoptosis 2006 11:773.

Neutral sphingomyelinase-induced ceramide triggers germinal vesicle breakdown and apoptosis in Xenopus laevis oocytes

P8
O. Coll, C. García-Ruiz, J. C. Fernández-Checa,.
Liver Unit, Hospital Clinic, IDIBAPS, IIBB-CSIC, Barcelona, Spain.

Sphingolipids, in particular ceramide, are dynamic regulators of many cellular processes, including cell growth, differentiation, apoptosis, and inflammatory responses. These diverse modes of action likely reflect that ceramide acts at different targets determined upon the site and/or mechanism mediating ceramide generation. For instance intracellular ceramide generation in cells transfected with neutral sphingomyelinase (NSMase) has been shown to result in apoptosis as opposed to when cells were exposed to exogenous NSMase. In addition, previous studies have indicated that NSMase triggered meiotic cell cycle progression in Xenopus laevis oocytes. Thus, our aim was to compare the effect of incubation vs microinjection of SMase from Bacillus cereus(bNSMase) on the maturation or apoptosis in Xenopus laevis oocytes. We show that while incubation of mature stage VI oocytes with bNSMase induces maturation measured as the germinal vesicle breakdown, its microinjection resulted in apoptosis, characterized by reactive oxygen species generation, GSH depletion, release of cytochrome c and Smac/Diablo from mitochondria, and caspase-3 activation. Preincubation of oocytes with with reduced GSH- ethyl ester prior to bNSMase microinjection, prevented these events and protected oocytes from bSMase-induced death. Taken together, these findings show a divergent action of bNSMase-induced ceramide on oocyte maturation or apoptosis depending on the site where ceramide is generated.

Effect of protein kinase C inhibitors on tumour cells via cellular and nuclear sphingomyelinase.

P9
E. Damaskopoulou, G. Cascianelli, I. Bernardini, M. P. Viola Magni, E. Albi.
Department of Clinical and Experimental Medicine, Physiopathology, University of Perugia, Italy.

Diacylglycerol (DAG), 3-phosphoinositides and ceramide acted on protein kinase C (PKC) which partecipated to signal transduction in many cells and mediated a number of intracellular functions1. Twelve different isoforms of PKC(α, βI, βII, γ, δ, ε, ζ, η, θ, ι, λ and μ) have been characterised. The atypical PKC ζ, ι and λ activity depended on phospholipids, such as ceramide and phosphatidylinositol 3,4,5 triphosphate, and not on DAG, phorbol ester and Ca++. PKC ζ activation ceramide- dependent stimulated cell proliferation. In order to study the effect of PKC ζ inhibition on DNA/RNA synthesis and on sphingomyelinase (SMase) activity, hepatoma cells were cultured in the presence of 9 different specific inhibitors for 48 hours. 3H-tymidine or 3H- uridine were added to the culture medium 3 hours before. The results showed that all inhibitors reduced the 3H-tymidine incorporation about40%-50%. 3H- uridine incorporation was reduced in the cells of 5-20% by all inhibitors except inhibitor I-1 and I-4 which induced a reduction of about 35%and in the nuclei of 35%-40% by all inhibitors except I-3 which had very little effect and I-2 which determined a reduction of 56%. The activity of cellular SMase was reduced 19-29% whereas the nuclear SMase activity was reduced about 45%- 70% by all inhibitors except I-9 which reduced the activity of 84%. It can be concluded that the inhibitors of PKC ζ induce a reduction of DNA and nuclear RNA synthesis accompanied by a strong inhibition of nuclear SMase.
1Musashi M et al., Int J Hematol 2000 72:12

Effect of aminocyclitols on mutant acid β-glucosidase activity in Gaucher disease fibroblasts. Their potential role as chemical chaperones.

P10
J. Duque¹, M. Lluch¹, G. Sánchez-Ollé², M. Egido-Gabás³, J. Casas³, A. Delgado^3,4, A. Llebaria³, D. Grinberg², L. Vilageliu², A. Chabás¹.
¹Institut de Bioquímica Clínica, Hospital Clínic. Barcelona, ²Departament de Genètica, Facultat de Biología, Universitat de Barcelona, ³Research Unit on BioActive Molecules, Department of Biological Organic Chemistry, IIQAB, CSIC. Barcelona. ⁴University of Barcelona, Faculty of Pharmacy, Unit of Pharmaceutical Chemistry (Associated Unit to CSIC), Barcelona.

Chemical chaperone therapy has been developed recently for several sphingolipidoses, including Gaucher disease. In this disorder, mutation N370S is the most prevalent one associated with important residual acid β- Glucosidase (β-Glu) activity, and different iminosugars has been reported to increase the mutant enzyme activity when added to patient fibroblast culture medium. We have studied the effect of several 2.5-60 μM aminocyclitols (C10, C9, C8, C4-PhBu derivatives of 1-deoxy-1-alkyl-amino inositol) on β-Gluactivity in fibroblast cultures derived from homozygous N370S patients. These compounds were potent inhibitors of fibroblast enzyme activity in vitro. For comparison, the effect of addition of the iminosugar N-Nonyl-DNJ (N-NDNJ)to WT and mutant fibroblast cultures was also carried out. N-NDNJ 5-20μMmarkedly increased the N370S mutant enzyme activity in both intact and lysed cells (1.5-2.2-fold), confirming previous reports with this iminosugar. WT and N370S patient fibroblast cultures were incubated for 4-6 days with aminocyclitols and β-Glu activity measured in intact and lysed cells. C10 andC4-PhBu (2.5-40 μM) inhibit enzyme activity in WT cells (27% with C10 30 μM; 50% with C4-PhBu 40 μM). In contrast, the mutant N370S enzyme activity is not affected or slightly stimulated with both compounds. The effect of C9 is similar in both WT and mutant cells. In the concentration range 2.5-60 μM, enzyme activity is moderately inhibited (33-55% with 40μM). The addition of C8 (2.5-60 μM) does not affect the WT enzyme, despite their inhibitory effect observed previously in vitro. The mutant enzyme activity is not affected or slightly increased (1.2–fold) at high C8 concentration.
Studies under different experimental conditions are needed for C10 and C4- PhBu to confirm the effect of these aminocyclitols on the N370S mutant activity fibroblasts.

New aminocyclitol libraries as candidates to chemical chaperone therapy for Gaucher Disease.

P11
M. Egido-Gabás¹, P. Serrano^1,2, J. Casas¹, M. Zucco³, G. Emeric³, A. Llebaria¹, A. Delgado^1,2.
¹Research Unit on Bioactive Molecules (RUBAM); Department of Organic and Biological Chemistry, Chemical and Environmental Research Institute of Barcelona (IIQAB-CSIC); Jordi Girona 18-26, 08034 Barcelona, Spain. ²University of Barcelona, Faculty of Pharmacy, Unit of Pharmaceutical Chemistry (Associated Unit to CSIC), Juan XXIII, s/n, 08028, Barcelona, Spain. ³ BayerCropScience GmbH Chemistry Frankfurt, Industriepark Höchst, G836. Frankfurt am Main, Germany.

Lysosomal storage diseases are a group of rare human disorders. One of them is Gaucher disease, a sphingolipidosis caused by a marked decreased in glucocerebrosidase (EC 3.2.1.45) activity. This deficiency cause to a progressive accumulation of glucosylceramide in macrophages, leading to hepatosplenomegaly, anemia, skeletal lesions, sometimes involving the central nervous system. The current therapeutic strategies to treat Gaucher disease consist of enzyme replacement therapy (ERT) or substrate reduction therapy (SRT). However, they are expensive and relatively ineffective for therapies involving the CNS. The molecular therapeutic strategy or chemical chaperone therapy for genetic metabolic diseases seems a promising alternative to restore the activity of mutant glucocerebrosidase.1-4
We present libraries of scyllo and racemic chiro N-substituted aminocyclitol derivatives. All compounds were synthesized by parallel solution-phasemethodology with the assistance of robotic technology. Chemical diversity were introduced by reaction of selected scaffolds with a set of aldehydes, acyl chlorides, sulfonyl chlorides, chloroformates, and amines to afford the corresponding amines, amides, sulfonamides, carbamates and ureas, respectively.5
Selected compounds were evaluated as inhibitors for recombinant glucocerebrosidase (Imiglucerase, Cerezyme®, Genzyme)6 with Ki values in the low micromolar range for the most active members. In conclusion, these compounds could become candidates for chemical chaperone therapy of Gaucher disease.
1Ishii et al. Bioch. Biophys. Acta 2004 1690:250. 2Matsuda et al. Proc. Natl. Acad. Sci. U S A 2003 100:15912. 3Sawkar et al. Proc. Natl. Acad. Sci. U S A. 2002 99:15428. 4Lin et al. Biochim. Biophys. Acta 2004 1689:19. 5J. Comb. Chem. In press. 6A generous gift of Cerezyme® from Genzyme Corporation is gratefully acknowledged.

Effects of ceramide 1-phosphate and its synthetic analogues on inflammatory pathways like stimulation of acid sphingomyelinase and phospholipase A2 in bone marrow derived macrophages.

P12
P. Gangoiti¹, S. Grijalvo^2,3, M. González¹, M. Granado¹, A. Delgado^2,3, J. Casas², A. Gómez-Muñoz¹.
¹Department of Biochemistry and Molecular Biology, School of Science and Technology, University of the Basque Country 48080 Bilbao, Spain,²Department of Biological-Organic Chemistry (IIQAB-CSIC), Barcelona, Spain and ³Unit of Pharmaceutical Chemistry, School of Pharmacy, University of Barcelona, Avda. Joan XXIII s/n; 08029 Barcelona, Spain.

The stimulation of acid sphingomyelinase (A-SMase) and the subsequent formation of ceramides are events that are associated to the pro- inflammatory actions of a variety of agonists including platellet activating factor (PAF). Therefore, there is increasing interest in biomedicine to develop drugs or compounds that are able to inhibit A-SMase activity in the absence of unwanted side effects. In this concern, we recently discovered that ceramide 1-phosphate (C1P) is a potent inhibitor of A-SMase both in intact macrophages as well as in cell free-systems. However, this putativeanti-inflammatory effect of C1P may be overcome by its ability to also activate the generation of arachidonic acid (through activation of PLA2) and the subsequent synthesis of eicosanoids, an action that is pro-inflammatory.In the present work, we have developed specific C1P analogs that are able to inhibit A-SMase activity in primary cultures of bone marrow-derivedmacrophages in the absence of PLA2 activation.
This study was supported by grants 040732 from Fundació la Marató de TV3, Barcelona (Spain), and 9/UPV 00042.310-15852/2004 from “Universidad del País Vasco” (UPV/EHU) (Bilbao, Basque Country).

Sphingosine-1-phosphate effects on the gel-fluid and lamellar-hexagonaltransitions of aqueous phospholipid dispersions.

P13
M. García-Pacios, M. I. Collado, M. A. Requero, A. Alonso, J. L. R. Arrondo, F. M. Goñi.
Unidad de Biofísica (CSIC-UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain.

Sphingosine-1-phosphate (SPP) has been recognized as a signalling molecule in cell metabolism. We have studied the effect of increasing proportions of SPP in aqueous phospholipid dispersions, using differential scanning calorimetry and infrared spectroscopy. The glycerophospholipids were either dielaidoylphosphatidyletanolamine (DEPE) or deuterated dipalmitoylphosphatidylcholine (d62-DPPC). SPP modified the gel-fluidtransition of both glycerophospholipids, shifting it to lower temperatures, and decreasing the reansition enthalpy. IR spectroscopy of natural SPP mixed with deuterated DPPC allows the independent observation of transitions in each mole. The DPPC CO group stretching vibration was shifted to lower frequencies, by about 2 cm-1, in the presence equimolar SPP. Pure SPP displayed a broad landothemic transition at ca. 65º C, that was accompanied by an increased lipid order. This transition was not detected in equimolar SPP: d62-DPPC mixtures. Finally, SPP was found to have a notorious effect on the lamellar-to-inverted hexagonal transition of DEPE, increasing the transition temperature and decreasing the transition enthalpy, i.e. stabilizing the lamellar vs. the inverted hexagonal phase.

Serum removal causes apoptosis in cultured alveolar macrophage through de novo syntesis of ceramide. Inhibition by ceramide 1-phosphate.

P14
M. Granado, P. Gangoiti, A. Gómez-Muñoz.
Department of Biochemistry and Molecular Biology, School of Science and Technology, University of the Basque Country, 48080 Bilbao, Spain.

Macrophages are involved in developing of pulmonary inflammation and parenchymal damage that occur in chronic obstructive pulmonary disease (COPD). The fundamental observation of this report is that incubation of alveolar macrophages NR8383 in the absence of serum leads to cell death. Serum removal also produced a concomitant increase in ceramide levels, which are detrimental for cells, and are usually implicated in the induction of apoptosis. In fact, incubation of the macrophages in the absence of serum caused phosphatidylserine exposure, which is a marker of apoptosis. Ceramide generation was mainly caused by de novo synthesis, as determined by inhibition of the incorporation of [3H]-palmitate into ceramides in the presence of myriocin, a selective inhibitor of serinepalmitoyltransferase. Another key observation of this work was that ceramide 1-phosphateinhibited the generation of the de novo synthesized ceramides, thereby preventing the macrophages from entering apoptosis.

Dependence of sphingoid bases concentration on growth phase and addition of zeolite in brewer’s yeast.

P15
I. Karmelic¹, F. Ivušic², S. Ribar¹, V. Maric², M. Mesaric¹.
¹Department of Chemistry and Biochemistry, School of Medicine and ²Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia.

Sphingolipids having a long-chain sphingoid base backbone are primarily located in the yeast’s plasma membrane. The two types of sphingoid bases in yeasts are sphinganine and its 4-hydroxy derivative, phytosphingosine, containing 16, 18 or 20 carbons. Sphinganine, phytosphingosine and ceramide, which are precursors of sphingolipids are involved in membrane signalling, regulation of cell wall biosynthesis, phospholipid biosynthesis and binding of cell surface glycoproteins. In addition, they are proven to play important roles in signal transduction during heat stress response, regulation of calcium homeostasis or components in calcium-mediate signalling pathways and in regulation of the cell cycle. Since little is known about the regulation of de novo synthesis of complex sphingolipids in any organism, an investigation into the regulation of the sphingoid bases’ biosynthesis in yeast may shed some light on this issue. The aim of our research was to investigate the effect of growth phase on the concentration and composition of sphingoid bases from brewer’s yeast. The second aspect of our research was to investigate the effect of natural zeolite clinoptilolite on sphingolipid metabolism in brewer’s yeast. The yeast cells were cultured in semi-industrialbioreactors in growth medium unhopped worth and in zeolite-supplementgrowth medium (1% zeolite into basic medium). Total sphingoid bases were extracted according to Riley at al. O-Phthaladehyde derivatives were analysed using reversed-phase high performance liquid chromatography (HPLC). The results point to the following conclusions: brewer’s yeast is a good source of sphingolipids and the predominant sphingoid base is phytosphingosine; the level of total sphingoid bases in brewer’s yeast changes depending on culture growth phase; the most obvious effect of the growth phase on the sphingoid base production has been observed in the case of phytosphingosine, the concentration of which is highest in the exponential phase; natural zeolite clinoptilolite increased the concentration of pytosphingosine which may indicate that zeolite acted at the level of phytosphingosine formation.

Ceramide formation by sphingomyelinases in red blood cells.

P16
D. J. López¹, L.-R. Montes¹, L. A. Bagatolli², F. M. Goñi¹, A. Alonso¹.
¹Unidad de Biofísica (CSIC-UPV/EHU), P.O. Box 644, E-48080 Bilbao, Spain and ²Memphys Center for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

Red blood cell membranes are particularly rich in sphingomyelin, thus they are potentially good substrates for sphingomyelinases. In this communication we summarize some recent results obtained with either an intrinsic sphingomyelinase activity or with an externally added (bacterial) enzyme. A latent intrinsic sphingomyelinase activity can be elicited by subjecting the erythrocytes to a hypertonic shock. This activity is magnesium–dependent,and its maximum value is reached 3-6 min after establishing the hypertonic conditions. The specific activity is 0.1 nmol/min x mg protein. When erythrocytes are stored at 4ºC, the activity remains intact for the first 48 h, then decreases. The intrinsic sphingomyelinase activity isdetergent–sensitive, increasing by ≈ 5-fold in the presence of sodium dodecylsulphate at subsolubilizing concentrations. Concominant measurements of intrinsic sphingomyelinase activity and ceramide concentrations reveal that the latter remain constant with time, indicating rapid conversion of the newly formed ceramide into other sphingolipids. Moreover, erythrocyte treatment with a bacterial sphingomyelinase induces rapid shape changes and generation of ceramide-rich domains in the red blood cell membranes.

Acidic sphingomyelinase is required for TNF-induced mitochondrial permeabilization and hepatocellular death via cardiolipin peroxidation.

P17
M. Marí, A. Colell, C. García-Ruiz, J. C. Fernández-Checa.
Liver Unit, Hospital Clinic, IDIBAPS, IIBB-CSIC; Barcelona, Spain.

TNF-α apoptotic signaling is a complex process that involves protein-proteininteractions and the participation of several intermediates. Sphingolipid generation through sphingomyelinase activation has been involved in apoptotic pathways induced by death ligands. In previous studies we have observed that the intrinsic resistance of hepatocytes to TNF is overcome by selectively depleting mitochondrial glutathione (mGSH) with apoptotic/necrotic hepatocyte death, and that acidic sphingomyelinase (ASMase) is necessary for efficient TNF-α–mediated hepatocellular damage.1Thus our aim was to examine the signaling in wild type and ASMase knockout hepatocytes responsible for the different outcome observed after TNF challenge following mGSH depletion. Cultured hepatocytes from 8-10 week- old wild type and ASMase KO mice (C57BL/6) were obtained by collagenase perfusion. Mouse hepatocytes were depleted of mGSH by (R/S)-3-hydroxy-4-pentenoate (HP, 0.5mM). Survival and chromatin morphology were assessed by GST release and Hoechst/PI staining. Bax, tBid and pBim translocation to mitochondria, JNK phosphorylation, release of cathepsin B, and caspase-3activation were determined by western blot. Mitochondrial membrane permeabilization (MMP) was assessed by the distribution of calcein/TMRM. Cardiolipin levels were analyzed by HPLC. ROS generation was analyzed fluorimetrically. The signaling events upstream of mitochondria such as Bax translocation to mitochondria, Bid truncation, NF-κB activation and transient JNK phosphorylation, were preserved in ASMase-deficient hepatocytes regardless of mGSH depletion. Only wild type mGSH-depleted cells underwent MMP with lower intact cardiolipin levels (40-50%) due to enhanced cardiolipin hydroperoxides content, cyt c release into the cytosol and caspase 3 activation followed by cell death. None of these features were observed inmGSH-depleted ASMase knockout cell. Interestingly, an early burst of ROS (beginning 15-30 min after TNF addition) was also only observed in mGSH- depleted wild type hepatocytes, and not in TNF-alone treated hepatocytes, nor in ASMase knockout hepatocytes despite mGSH depletion. Furthermore, in previous studies we have monitored the cellular distribution of ganglioside GD3 during TNF-α signaling indicating its mitochondrial targeting (as soon as 30 min after TNF addition) in wild type but not in ASMase knockout hepatocytes, suggesting that ASMase is necessary for this event. These suggest that mGSH modulates hepatocellular sensitivity to TNF through control of mitochondrial ROS generation targeting mitochondrial cardiolipin status.
1García- Ruiz et al., J. Clin. Invest. 2003 111:197; Marí et al., J. Clin. Invest. 2004 113:895.

Involvement of nuclear sphingomyelinase in embryonic hippocampal cell differentiation vitamin D3-induced.

P18
F. Marini¹, E. Bartoccini¹, G. Cascianelli¹, M. García-Gil², M. P. Viola Magni¹, E. Albi¹.
¹Department of Clinical and Experimental Medicine, Physiopathology, Policlinico Monteluce, 06100 Perugia, Italy and ²Department of Physiology and Biochemistry, University of Pisa, Italy.

It is known that vitamin D3 induces cell differentiation by activating neutral sphingomyelinase (N-SMase). Recently it has been demonstrated that nuclearN-SMase rather that cellular N-SMase is involved in the early signal transduction after cell stimulation to proliferation and/or differentiation 1. The aim of the present work was to study the N-SMase activity modifications in the differentiation of embryonic hippocampal cells induced by vitamin D3 treatment. At this end HN9.10e, which exhibited morphologic and trophic activity typical of primary hippocampal neurons, were used. Vitamin D3 was added to culture medium at the final concentration from 50 to 400 nM and the morphological differentiation and nuclear N-SMase activity were evaluated. The results showed that 50-100 nM was the optimal concentration to obtain cell differentiation whereas 400nM concentration induced the cells to apoptosis. In fact after 50-100nM vitamin D3 treatment, the cells showed the lengthening of the soma while neurite and dendrites appeared after 5 days of culture. Bcl2 expression increased 16 times higher respect to the control and also NGF resulted strongly increased. Only 2 hours of treatment, the nuclear N-SMase activity increased of two time and 14C palmitic acid incorporation in lipids showed an increase of ceramide production. The results suggested the involvement of nuclear SM pathway in HN9.10 differentiation.
1 Albi E and Viola Magni MP Biology of the Cell 2004 96:657.

A new family of dihydroceramide desaturase inhibitors.

P19
X. Matabosch^1*, J. M. Muñoz-Olaya^1*, J. Casas¹, A. Delgado^1,2, A. Llebaria¹, G. Fabriàs¹.
¹Research Unit on BioActive Molecules, Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18, 08034-Barcelona, Spain and ²Unit of Pharmaceutical Chemistry, School of Pharmacy, University of Barcelona, Avda. Joan XXIII s/n; 08029 Barcelona, Spain.

The last step in the de novo biosynthetic pathway of ceramide involves the introduction of the (E)-4 double bond in dihydroceramide by dihydroceramide desaturase (DHCD). The only inhibitors of this enzyme so far described include a family of cyclopropene analogs of ceramide (GT11 and analogs). By means of a novel procedure to determine DHCD activity, a new family of DHCD inhibitors has been identified among a library of dihydroceramide analogs. The biochemical assay utilizesN-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-erythro-sphinganine as substrate and aHPLC-fluorimetric method, and can be used both in vitro and cultured cells. From a structural standpoint, the inhibitors are analogs of dihydroceramide in which a specific methylene group of the long chain base has been replaced by an heteroatom. The several compounds differ in both the heteroatom substituents and the N-acyl group. Their preliminary activities in both rat liver microsomes and mammalian cells are reported.
*Both authors contributed equally to this work and are listed in alphabetical order.

Analysis of epidermal lipids in dogs. A preparative study for validation of an animal model of atopic dermatitis.

P21
I. Popa¹, A. Piekutowska¹, J. Chapman¹, H. Gatto², D. Pin³, M. Haftek¹, J. Portoukalian¹.
¹University Lyon-I, Dermatology, Edouard Herriot Hospital 69437 Lyon Cx 3, France. ²Virbac, Carros, France. 3Ecole Nationale Vétérinaire de Lyon, Marcy l’Etoile, 69280, France.

Atopic dermatitis (AD) is a well characterized clinical entity in dogs. We have observed ultrastructural changes in stratum corneum (SC) morphology and lamellar lipid organization in the non-lesional skin of atopic dogs using osmium and ruthenium tetroxide postfixation. Because of the observed clinical and morphological similarities between the human and dog atopic skin, we speculated that biochemical modifications concerning SC lipid expression described in man could also underlie AD in dogs. Dog SC was taken using strips. Ten consecutive strips were needed to reach the viable epidermis. We purified and analyzed individually for each strip the different classes of free lipids (free fatty acids, ceramides, phospholipids, glycolipids, gangliosides) and protein-bound lipids (ceramides and fatty acids) and compare to the lipid profile of human epidermis. This comparison showed us that dog epidermis contain similar ceramides species as in humans with the same three sphingoid bases coupled to long-chain fatty acids, especially toomega-hydroxylated fatty acids. In the case of human AD, it has been reported that the deficiency in acylceramides was correlated to the defects in skin barrier. We have compared the lipid profiles of normal dog epidermis with those of atopic dermatitis dogs, and found that the epidermis of atopic dogs have a much lower lipid content with a marked deficit in ceramides. A detailed knowledge about the lipid composition of normal SC in dogs is an indispensable step towards the biochemical characterization of canine AD and should help to define an animal model of this disease.

Sphingoid bases and gangliosides in infant formulas.

P22
S. Ribar¹, I. Karmelic¹, D. Ivankovic², M. Mesaric¹.
¹Department of Chemistry and Biochemistry, School of Medicine, University of Zagreb, Šalata 3, 10000 Zagreb, Croatia. ²School of Medicine, University of Zagreb, Šalata 3, 10000 Zagreb, Croatia.

Sphingolipids are a group of lipids present in all eukaryotic cells. They consist of a long-chain sphingoid base as the backbone. The prevalent long-chainbases of most mammals are D-erythro-sphinganine and sphingosine. Gangliosides comprise a group of glycosphingolipids that contain one or more sialic acid moieties. They are one of the components of human milk that have been demonstrated to play important roles in neuronal development, neuropathologic processes, and receptor functions with respect to protein hormones, interferon, fibronection and bacterial toxins. The aim of our research was to establish if there are the diferences in sphingoid bases concentrations between human milk and infant formulas. The second aspect of the research was to ascertain whether there was any difference in the concentrations of gangliosides between human milk and infant formulas. Sphingolipids were extracted from human milk and infant formulas. Free and total sphingoid bases were obtained by base and acid hydrolysis respectively. Sphingosine and sphinganine were derivatized with the OPA (O- Phtaldialdehyde) reagent and analysed by high performance liquid chromatography. After sphingolipid extraction, gangliosides were quantitatively determined in supernatant with a colorimetric resorcinol- hydrochloric acid method. The results of this research indicate the significant differences between the concentrations of sphingoid bases and gangliosides in infant formulas and human milk. Based on the obtained results, it can be concluded that despite all efforts made to produce infant formulas as similar to human milk as possible, in terms of their structure and the amount of their constituents, there are differences that could be biologically significant and thus need to be further researched.

Preliminary study of the chaperone effect on mutated glucocerebrosidases as a treatment for Gaucher disease.

P23
G. Sánchez-Ollé¹, M. Egido-Gabás², J. Duque³, J. Casas², A. Chabás³, L. Vilageliu¹, D. Grinberg¹.
¹Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, ²RUBAM (Research Unit on BioActive Molecules), Departament de Química Orgànica Biològica, IIQAB-CSIC, Barcelona and ³Institut de Bioquímica Clínica, Corporació Sanitària Clínic, Barcelona.

Gaucher disease (GD) is a glycosphingolipid storage disorder caused by deficiencies in lysosomal acid β-glucosidase (GBA), resulting in progressive accumulation of glucosylceramide in macrophages of liver, spleen, bone marrow and, sometimes, central nervous system. It has been reported that some iminosugar compounds are able to work as chemical chaperones, at subinhibitor concentrations, and increase the activity of wild-type and mutated GBAs.
The aim of this work is to express and characterize mutant GBA alleles and assay the chaperone effect of the iminosugars N-(n-nonyl)deoxynojirimycin(NN-DNJ) and N-(n-butyl)deoxyno-jirimycin (NB-DNJ) on the expressed proteins. Eleven different mutated alleles have been studied. COS-7 cells have been transfected with the pcDNA3 expression vector carrying the wild- type or the mutated cDNAs. The residual enzymatic activities, given as percentage of the wild-type enzyme activity are the following: N188S: 53.84%, G202R: 24.84%, H255Q: 77.09%, E326K: 30.51%, N370S: 8.11%, G377S: 11.06%, I402T: 19.37%, D409H: 16.46%, L444P: 2.42%, [N188S;E326K]: 12.78% and [H255Q;D409H]: 16.05%.
Stable transfected cells, selected with geneticin, were treated for 6 days with NN-DNJ (2.5 and 5 μM) or NB-DNJ (5 and 10 μM). Preliminary results show an increase of the enzymatic activity for the wild-type enzyme (at 2.5 and 5 μM) and for the mutated N188S (at 2.5 μM) and [N188S;E326K] (at 5 μM) GBAs. Surprisingly, a decrease in protein activity was also observed for the N188S (at 5 μM) and [N188S;E326K] (at 2.5 μM) mutated enzymes. After treating the cells with NB-DNJ, the increase in activity was observed for the wild type enzyme (at 10 μM), but also for the N188S (at 5 and 10 μM), N370S (at 10 μM) and [N188S;E326K] (at 10 μM) mutated proteins.
After these promising preliminary results, new compounds (aminociclitol derivatives), will be assayed.

Localization of sphingosine kinase-1 in detergent-resistant membranes of C2C12 myoblasts.

P24
E. Sarchielli¹, C. Sassoli², L. Becciolini¹, L. Formigli^2,4, C.Donati^1,4, G. Nemoz³, P. Bruni^1,4, E. Meacci^1,4.
Departments of ¹Biochemical Sciences, ²Anatomy, Histology and Forensic Medicine, University of Florence, ³Laboratoire de Physiopathologie des Lipides et Membranes, Institut National des Sciences Appliquees de Lyon, Villeurbanne, France. ⁴Interuniversity Institute of Myology (IIM), Italy.

Sphingosine kinase (SphK), which catalyzes the formation of sphingosine 1- phosphate starting from sphingosine and ATP, has been recognized to be critical for the control of cell proliferation, differentiation and motility in a large number of cells as well as in C2C12 myoblasts1,2. Recently, we demonstrated that SphK activity as well as SphK1 protein content are increased upon the attainment of the cellular confluence and the terminal differentiation in myotubes, consistently with an important role of the enzyme in cell growth arrest and myogenesis3. Although in many circumstances the major enzymatic isoform, SphK1, is largely localized in the cytoplasm, it is generally accepted that SphK functionality requires membrane localization.
The study of membrane localization of SphK1 in C2C12 myoblasts revealed that the enzymatic activity increase observed in confluent myoblasts, was paralleled by a significant rise of membrane-associated enzyme and the its enrichment in detergent-resistant membranes, as indicated by the enhancedco-localization with specific lipid raft markers such as cholera toxin B subunit and co-immunoprecipitation with caveolin-1, the caveolae scaffold protein.
Given that, phosphatidic acid (PA), generated by phospholipase D, has been proposed to direct SphK1 membrane translocation4, we have investigated the possible role of PLD/PA system in the recruitment of SphK1 in these membrane compartments. Interestingly, [3H]PA locally increased when the cells reached the confluence and co-localized with SphK1 in fully confluent myoblasts as judged by confocal microscopy analysis. Interestingly, cell treatment with PLD1-siRNA or 1-butanol drastically reduced PA formation, SphK1 detergent-resistant membrane activity and association, while the overexpression of PLD1 augmented SphK activity as well as SphK1 protein content in that membrane compartment. Both experimental conditions also greatly affected cell growth and myogenic differentiation.
In conclusion, these data support that PLD/PA system play an important role in SphK1 translocation to detergent-resistant membranes of confluent myoblasts and represent a crucial event in C2C12 myoblast growth and myogenesis
This work was supported in part by funds from Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR-PRIN 2003 to P.B.; MIUR-PRIN 2004 to E.M.), University of Florence (ex- 60%) to P.B. and E.M., Ente Cassa di Risparmio di Pistoia e Pescia to E.M.
1Liu et al., Prog Nucleic Acid Res Mol Biol. 2002 71:493. 2Donati et al., FASEB J. 2005 19, 449.3Meacci et al. (2006) submitted. 4Delon et al., J. Biol. Chem. 2004 279:44763.

Resveratrol induces apoptosis in human melanoma A375 cells by ceramide signalling.

P25
P. Signorelli¹, C. A. La Porta², F. Scarlatti¹, R. Maffei¹, G. Sala¹, R. Ghidoni¹.
¹Laboratory of Biochemistry and Molecular Biology, San Paolo University Hospital and ²Department of Biomolecular Science and Biotechnology, University of Milan. Milan, Italy.

Resveratrol is a plant-derived antioxidant and a phytoestrogen polyphenol that has been extensively studied in vivo and in cultured cells lines for its antitumoral activity. The aim of this study was to investigate the effects of resveratrol on the growth and survival of human melanoma A375 cells and to identify the mechanism of resveratrol action in this model. Resveratrol reduced proliferation and viability and increased the intracellular concentration of the pro-apoptotic sphingolipid mediator ceramide. The increase in ceramide was due to both its de novo synthesis and to sphingomyelin hydrolysis since it was reduced by myriocin, an inhibitor ofserine-palmitoyl-transferase, and completely impaired by glutathione, an inhibitor of neutral sphingomyelinase. The abrogation of ceramide increase by glutathione but not its reduction by myriocin rescued cells from the loss of viability induced by resveratrol. Moreover resveratrol induced cell death was related to the cleavage of poly-(ADP-ribose)-polymerase and this event was prevented by glutathione and not by myriocin.
We can conclude that resveratrol triggered ceramide signalling in human melanoma A375 cells and the pool of ceramide derived from neutral sphingomyelinase activity was required for PARP cleavage and for apoptosis induction.

The role of sphingolipids, glycolipids and phospholipids in the Drosophila epithelial and glial permeability barriers.

P26
M. Strigini¹ and D. Karagogeos^1,2.
¹IMBB/FORTH, Iraklio, Crete, Greece and ²Medical School, University of Crete, Iraklio, Crete, Greece.

Cell adhesion depends on specialised structures, such as occluding and adherens junctions. These junctions localize to specific membrane regions, for example to distinct domains along the apical-basal axis in polarised cells. Indeed, junction formation and cell polarization are interdependent. Polarised cells compartmentalize proteins as well as lipids in their membranes. While much progress has been made in learning how several junction proteins contribute to cell polarity and how they get localised, little is known about the contribution of lipids to cell polarization and cell adhesion.
Drosophila is an attractive model system to study in vivo the genetic, molecular and cellular basis of developmental and cell biological processes. We try to exploit such system to study the role of lipids (and in particular sphingolipids, glycosphingolipids, and phospholipids) in the establishment of cell polarity and cell adhesion. We concentrate on the formation of septate junctions (SJ), a type of occluding junctions found among epithelial cells and among glial cells that enwrap the nervous system. SJ sustain the epithelial and glial permeability diffusion barriers. Several proteins are known to localise to SJ, and their role in the establishment and maintenance of SJ has been investigated by many groups, including ours. We have now turned our attention to lipids.
We have just embarked in the characterization of the subcellular distribution of different lipid subtypes in the polarised cells of Drosophila epithelia. In addition, as a handle to the genetic study of lipids in this context, we investigate the contribution of lipid metabolism genes to the process of SJ formation. We are screening mutants in sphingolipid, glycosphingolipid, and phospholipid metabolism genes using established dye penetration assays, to probe the permeability of the epithelial and glial barriers. We will then proceed to assess the molecular, cellular and structural defects in the mutants with disrupted barriers, by looking at the organization of SJ and the morphogenesis of the glia forming the so called blood-brain barrier. We will report on our preliminary results.

Quantitation and composition of ceramide-1-phosphate in mouse tissues.

P27
H. Van Overloop, P.P. Van Veldhoven.
Pharmacology, Katholieke Universiteit Leuven, Belgium.

Ceramide-1-phosphate (Cer-1-P) appears to be powerful bioactive sphingolipid, involved in different physiological processes, like mitosis, inflammation, phagocytosis and apoptosis. Until now, not much is known about the basal levels and composition of Cer-1-P in mammalian tissues and cells. To determine this sphingolipid in tissues/cells, we developed a method, based on the strong retention of phosphate esters on aminopropyl SPE columns, followed by elution and dephosphorylation with alkaline phosphatase, and rephosphorylation with recombinant ceramide kinase (CERK) in the presence of [γ-32P]-ATP. This protocol allows the quantification of Cer- 1-P in the low nmole range. When applied to mouse tissues, TLC separation of the 32P-labeled ceramides revealed at least three major species, differing in the N-acyl chain: long chain, very long chain and2-hydroxy (very) long chain fatty acids. Both non-hydroxy- and 2-hydroxyfatty acid containing Cer-1-P are most enriched in cerebellum, estimated at ~ 20 nmol and 8 nmol/g tissue, respectively. Lowest Cer-1-P levels are found in liver and kidney (~ 5 nmol/g).
This work was supported by the Flemish government (Geconcerteerde Onderzoeksacties GOA/2004/08), the Belgian Ministery of ‘Federaal Wetenschapsbeleid’ (Interuniversitaire Attractiepolen IAP-P5/05) and the Flemish ‘Fonds voor Wetenschappelijk Onderzoek’ (G.0405.02). H.V.O. is an aspirant from the Flemish ‘Fonds voor Wetenschappelijk Onderzoek’.

The extended family of neutral sphingomyelinases.

Hannun YA
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425

The levels of ceramide are under complex regulation by the action of a combination of enzymes whose own regulation serves to transduce the actions of stimuli into specific cell responses. Neutral(N-SMases) regulate the formation of ceramide through the hydrolysis of sphingomyelin substrates. A combination of genetic, informatic and biochemical approaches has led to the identification of a novel family of N-SMases including the yeast Isc1 and mammalian nSMase2. These enzymes share similarin vitro catalytic mechanisms and a similar requirement for anionic phospholipids. Yeast Isc1 is activated by mitochondrial anionic phospholipids, localizes to mitochondria, and participates in thepost-diauxic response of yeast cells. In human cells, nSMase2 is activated by cell-cell contact in a mechanism involving translocation to the plasma membrane. This enzyme appears to regulate the formation of specific very long chain ceramide species with suggested roles in mediating dephosphorylation of the retinoblastoma protein and the induction of cell cycle arrest in response tocell-cell contact and confluence. These results underscore the specific functions of individual pathways of ceramide metabolism in eukaryotic cell regulation.

Design and synthesis of acidic ceramidase inhibitors and their characterization using a novel fluorogenic substrate.

C Bedia¹, G Triola¹, S Grijalvo^1,2, J Casas¹, A Delgado^1,2, A Llebaria¹, T Levade³ and G Fabriàs¹.
¹Research Unit on BioActive Molecules (RUBAM), Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18. 08034-Barcelona. Spain. ²Departamento de Farmacología y Química Terapéutica, Facultad de Farmacia, Universidad de Barcelona, Unidad de Química Farmacéutica (Unidad Asociada al CSIC), Avda. Juan XXIII s/n. 08029- Barcelona. Spain. ³Laboratoire de Biochimie et Maladies Metaboliques, INSERM U466, CHU Rangueil. 1, avenue Jean Poulhes. 31059 Toulouse cedex. France.

The first step in the catabolic route of ceramide is its amide hydrolysis by ceramidases. Several types of ceramidases have been reported that differ in their optimum pH, intracellular location and substrate specificity. Mutations in the acidic form gives rise to Farber disease (1), a rare lysosomal storage disorder for which there is no current therapy. A novel strategy that has afforded promising results in Gaucher and Fabry diseases is the active site directed chaperone approach. This strategy relies on the use of competitive enzyme inhibitors at sub-inhibitory concentrations to aid the correct folding of the mutated enzyme, thus favouring its transport to the lysosome and increasing its residual activity. Although several inhibitors of ceramidases have been reported (2-4), potent inhibitors of the acidic enzyme are not available. The only acid ceramidase inhibitor so far described is N-oleoylethanolamine, and its potency is rather low (4). In contrast, potent and selective inhibitors of mechanistically similar hydrolases are known (5). Some of these inhibitors include anelectron-deficient carbonyl group that reacts with the active-site nucleophylic amino acid to form an hemiacetal intermediate, which mimics the reaction transition state thus inhibiting the hydrolysis

(6). Taking into account these studies and considering that N-acyl sphingosines with acyl chain lengths below C10 are poor substrates of the neutral ceramidase (7), two families of compounds were designed, synthesized and their activity as selective inhibitors of the acidic ceramidase was determined both in vitro and in cultured cells using a fluorogenic substrate in a 96-well plate format.

Table. Inhibition of the acidic ceramidase.

COMPOUND	IC50 (µM)	IC50 (µM)
	In vitro	In cell culture
GT85	83	1.33
GT98	77	12.3
GT99	28	0.81
GT102	210	0.65
GT103	23	3.41
GT104	55	5.15
GT109	255	3.29

Among the compounds tested, a family of ketoamides exhibited inhibitory activity of the acidic ceramidase, both in enzyme preparations in vitro and in cultured Farber cells that overexpress the acidic ceramidase (Table). Kinetic studies conducted with GT85 revealed that this compound is a competitive inhibitor with a Ki of 16 µM (Figure). These ketoamides, which probably behave as reversible transition state mimics, are the first inhibitors of the acid ceramidase with potencies in the low µM range. Remarkably, these compounds have a good selectivity as compared to the neutral ceramidase in vitro, which requires around 20 fold higher concentrations for inhibition. Thus, these ketoamides are attractive candidates as chemical chaperones of potential utility in the therapy of Farber disease.

Some of the ketoamides bear a cyclopropene ring in the long chain base and have dual inhibitory activity of both dihydroceramide desaturase and the acidic ceramidase. Besides their putative usefulness as chemical chaperones, these compounds also produce a decrease of intracellular ceramide and are thus interesting as substrate reduction agents for Farber disease treatment.

References
1. He X, Okino N, Dhami R, et al., J. Biol. Chem. 2003, 278, 32978-32986. 2. Bielawska A, Greenberg MS, Perry D, et al., J. Biol. Chem. 1996, 271, 12646-12654. 3. Selzner M, Bielawska A, Morse MA, et al., Cancer Res. 2001, 61, 1233- 1240. 4. Strelow A, Bernardo K, Adam-Klages S, et al., J. Exp. Med. 2000, 192, 601-612. 5. Boger DL, Sato H, Lerner AE, et al., Proc. Natl. Acad. Sci. USA. 2000, 97, 5044-5049. 6. Koutek, B, Prestwich, GD, Howlett, et al., J. Biol. Chem. 1994, 269, 22937-22940. 7. El Bawab, S, Usta, J, Roddy, et al., J. Lipid Res. 2002, 43, 141-148.

CD1 glycosphingolipid antigen synthesis for the study of protein-carbohydrate interactions.

Panza L,¹ D. Colombo,² F. Compostella,² L. Franchini² and F. Ronchetti^2
1DISCAFF, Università degli Studi del Piemonte Orientale, Novara, Italy
²Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina , Università di Milano, Via Saldini 50, 20133-Milano, Italy

Different classes of foreign and self lipid antigens are known to be presented by CD1 antigen presenting molecules to T cells.[1] Several studies suggest that CD1-restricted T cells perform a variety of important roles in cell-mediated immunity, although different responses have been observed after stimulation by foreign or self antigens. While the role of these proteins in bacterial infections can in general be considered clearly evaluated, their functions in the balance between the maintenance of tolerance and the development of autoimmune disease require further investigations.

Sulfatide (a mixture of 3-sulfated b-D-galactosylceramides with different fatty acids at the ceramide moiety) is one of the glycolipid antigens so far characterized that bind group I CD1 molecules.[2] Much interest is now addressed to the elucidation of the mechanism of antigen presentation. In this context a series of derivatives structurally related to sulfatide have been synthesized as pure compounds for biological assays to shed light on how differences in antigen structures can influence the activity or as probes for binding assays.

For the elucidation of the role of the lipidic moiety structure in antigen presentation, a family of sulfatides, constituents of the natural mixture and bearing palmitic, stearic, behenic or nervonic fatty acid chains, has been synthesized; these compounds have been shown to be capable of stimulating sulfatide-specific andCD1-restricted T-cell clones.[3]

In order to have a deeper knowledge of the various parameters affecting lipid binding to the different CD1 isoforms, a fluorescent sulfatide has been prepared, with a fluorophor attached on positon 6 of galactose, thus not interfering with the binding; the fluorescent probe binds to soluble recombinant human CD1a and is a valuable tool for the study of CD1 antigen binding properties.[4] Further biological tests will show if the activity of this derivative is maintained also with the other CD1 isoforms.

Finally, a b-D-galactosylceramide fully protected with selectively removable protecting groups has been synthesized; this derivative is a valuable scaffold which allows the access to a series of sulfatide related compounds, e.g. sulfated galactosylceramides differing in the position of the sulfate group on galactose for the study of the influence of the sulfate group position on T cells activation.

[1]Brigl, M.; Brenner, M. B. (2004) Annu. Rev. Immunol. 22, 817-890.
[2]Shamshiev, A.; Gober, H.-J.; Donda, A.; Mazorra, Z.; Mori, L.; De Libero, G. (2002) J. Exp. Med. 195, 1013-1021.
[3]Compostella F.; Franchini, L.; De Libero, G.; Palmisano, G.; Ronchetti, F.; Panza, L. (2002) Tetrahedron 58, 8703- 8708.
[4]Franchini L.; Compostella F.; Donda A.; Mori L.; Colombo D.; De Libero G.; Matto P.; Ronchetti F.; Panza L. (2004)

Eur. J. Org. Chem. 4755-4761.

Synthetic hydroxylated aryl-naphthalene derivatives as resveratrol rigid analogues showing ceramide-mediatedantiproliferative and pro-apoptotic properties.

Filippo Minutolo¹, Giusy Sala², Annalisa Bagnacani², Simone Bertini¹, Isabella Carboni¹, Giovanni Prota¹, Simona Rapposelli¹, Nicoletta Sacchi³, Marco Macchia¹, Riccardo Ghidoni^2
1Dept. Pharmaceutical Sciences, University of Pisa – ²Lab. Biochemistry & Molecular Biology, San Paolo University Hospital, Medical School, University of Milan – ³Dept. Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA

The cancer chemoprevention and the antiproliferative effect associated with dietary polyphenol resveratrol has prompted many research groups to devote their efforts in the search for new and more efficient analogues. The antiproliferative and proapoptotic properties of resveratrol have been shown to be due to an accumulation of endogenous ceramide, a pro-apoptotic natural sphingolipid, in cancer cell lines. In particular, the apoptotic cell death has been attributed more likely to the de novo synthesized ceramide, rather than to sphingomyelin hydrolysis. Moreover, the presence of phenolic hydroxy-groups have been demonstrated to be crucial in preserving this mechanism of action, since tri-O-methylated resveratrol, although showing potent growth inhibition properties in cancer cells, did not induce any accumulation of ceramide. The trans-configuration of the stilbene double bond of resveratrol also seems to be important in preserving the ceramide accumulation mediated pro-apoptotic properties.

Nevertheless, the trihydroxylated trans-stilbene chemical structure of resveratrol may undergo, as well as most stilbene derivatives, to several chemical and metabolic degradations. One well-known transformation is represented by the photochemical (E)/(Z)-isomerization of the double bond, to produce compounds of (Z)- configuration that are generally less active to inhibit cellular growth and promote apoptosis. The main metabolic reactions on stilbene derivatives involve oxidation reactions on the double bond and on theelectron-rich phenolic rings, producing highly reactive oxidized metabolites (e.g. epoxides, arene oxides, benzylic alcohols, etc.). These modifications may constitute a significant problem because in vitro biological data may be misinterpreted and, if these compounds are submitted to in vivo assays, there might be a concurrence of several metabolites to define the activity and toxicity profiles. For these reasons we decided to investigate the possibility of preparing resveratrol analogues possessing a more rigid and stable scaffold, by replacing the 4-hydroxy-styrene portion with a beta-naphthol portion. We then designed and synthesized a series of naphthalene-based resveratrol analogues, which possess either “all-free” hydroxyls, or various combinations of O-methylated OH substituents, to assess the importance of the phenolic groups also in this new class of resveratrol analogues.

The biological assays proved that the presence of a naphalene ring generally increases functional proapoptotic properties of the new molecules when compared to similarly substituted stilbene analogues. Furthermore, the concurrent presence of three hydroxyls on the phenyl-naphthalene core is still mandatory, as for resveratrol itself, to induce ceramide-mediated apoptotic cell death, since methoxy-substituted naphthalene derivatives, in spite of some good antiproliferative activity found, did not cause significant ceramide accumulation levels.

New aminocyclitols as candidates to chemical chaperone therapy for gaucher disease.

Egido-Gabás M¹, Duque J², Murillo A² ,Canals D¹, Delgado A^1,3, Llebaria A¹, Chabás A², Casas J^1
1Research Unit on BioActive Molecules (RUBAM), Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18.08034-Barcelona. Spain. ²Institut de Bioquímica Clínica, Corporació Sanitària Clínic, Mejia Lequerica, s/n. 08028-Barcelona. Spain. ³Departamento de Farmacología y Química Terapéutica, Unidad de Química Farmacéutica (Unidad Asociada al CSIC), Facultad de Farmacia, Universidad de Barcelona, Avda. Juan XXIII s/n. 08034-Barcelona. Spain.

Glycosphingolipids are amphipathic molecules that contain a hydrophobic moiety (ceramide) and a hydrophilic residue (sugar). They play an important role in cell recognition, proliferation and differentiation, immunorecognition and signal transduction1.

Glycosphingolipidoses are lysosomal storage diseases, a group of rare human disorders. One of them is Gaucher disease, a sphingolipidosis caused by a marked decreased in glucocerebrosidase (EC 3.2.1.45) GBA) activity. This deficiency leads to a progressive accumulation of glucosylceramide in macrophages, resulting in hepatosplenomegaly, anaemia, skeletal lesions and, sometimes, central nervous system (CNS) involvement.

The current therapeutic strategies to treat Gaucher disease involve enzyme replacement therapy (Imiglucerase, Cerezyme®, Genzyme) or substrate reduction therapy (Miglustat, Zavesca®, Actelion). However, they are expensive and relatively ineffective for CNS involvement cases2. The molecular therapeutic strategy for genetic metabolic diseases based on the use of chemical chaperones can be a promising alternative for restoration of mutant glucocerebrosidase activity3-4.

We present a new series of aminocyclitol derivatives that have been synthesized and tested as glucocerebrosidase inhibitors5. Some of them are potent and competitive inhibitors at low concentrations (Ki<3microM) of lysosomal GBA from rat liver and Imiglucerase (Cerezyme®, Genzyme6), and they are also active on the residual GBA activity present in fibroblasts of Gaucher disease patients with different genotypes (N370S/N370S, L444P/L444P, and N370S/L444P). These compounds are specific inhibitors of glucocerebrosidase since they are not inhibitors of other glucose-metabolizing enzymes (alpha-glucosidasefrom Baker’s Yeast, beta-glucosidase from Crude Almonds and microsomal glucosylceramide synthase from rat liver). They are also inactive against other lysosomal enzymes involved in ganglioside and glycosphingolipid metabolism, (beta-galactosidase, alpha-galactosidase A and N-acetyl-alpha-galactosaminidase from human fibroblasts).

Among the active compounds, three of them protect GBA from thermal denaturation. Consequently, these compounds might be considered as good candidates for a chemical chaperone therapy of Gaucher disease.

References
1. Kolter, T., Sandhoff, K. Angew. Chem. Int. Ed. 1999, 38, 1535-1568. 2. Germain, D.P., Clin. Genet., 2004, 65, 77- 86 3. Sawkar, A.R., Cheng, W., Beutler, E., Wong, C., Balch, W.E. and Kelly, J.W., PNAS, 2002, 99, 15428-15433. 4. Lin, H., Sugimoto, Y., Ohsaki, Y., Ninomiya, H., Oka, A., Taniguchi et al. Biochim. Biophys. Acta 2004, 1689, 19-228. 5.Egido-Gabás, M., Serrano, P., Casas, J., Llebaria, A., Delgado, A. Org. Biomol. Chem. 2005, 3, 1195-201. 6. A generous gift of Cerezyme® from Genzyme Corporation is gratefully acknowledged.

Membrane fusion induced by PlcHR2, a novel sphingomyelinase / phospholipase C from pseudomonas aeruginosa.

L.-Ruth Montes¹, Maitane Ibarguren¹, Félix M. Goñi¹, Michael L.Vasil² and Alicia Alonso^1
1Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), Universidad del País Vasco, 48080 Bilbao, Spain. ²Department of Microbiology, University of Colorado, Denver, CO 80262, USA.

PlcHR2 from P. aeruginosa is a heterodimeric complex, formed by PlcH, the subunit containing the sphingomyelinase/phospholipase C active center, and PlcR2, a chaperone that modulates the catalytic activity.

PlcHR2 is the paradigm enzyme of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial genera. In the present work we study the structural effects of PlcHR2 when acting on liposomal and cell membranes containing sphingomyelin together with other lipids. Both PlcHR2 and its separate components bind Ca2+ and, while this cation has no detectable effect on the hydrolytic activity, it inhibits PlcHR2 -induced haemolysis. In liposomes (large unilamellar vesicles) the enzyme complex inducesvesicle-vesicle fusion at similar rates, and to similar extents, in the presence and absence of Ca2+. However, this cation increases dramatically the rate of vesicle aggregation, and the rate and extent of enzyme-inducedrelease of vesicular aqueous contents (“leakage”).

Plasma membranes purified from rat liver are made mostly of detergent-resistant microdomains (lipid rafts).

Popa I¹, Bionda C¹, Ardail D.², Portoukalian J.^1
1Department. of Dermatology, Edouard Herriot Hospital,69437 Lyon Cx 03, France; ²Laboratory of Radiobiology, Lyon-Sud Medical School 69921 Oullins Cx, France

Plasma membranes are thought to be made of assembled detergent-resistant (rafts) and detergent-soluble(non-rafts) microdomains. However, most studies isolate lipid rafts from whole cells and very few investigations aim to determine the respective proportions of rafts and non-rafts domains in plasma membranes.

In the present study, the plasma membrane fraction of rat liver has been purified, then the raft and non-raftdomains have been isolated from the plasma membrane. Following solubilization of the membranes in TritonX-100 1%, fractions were separated by ultracentrifugation on Optiprep gradient. The specific marker proteins flottilin and glyceraldehyde-3 phosphate dehydrogenase (Gapdh) were monitored by western blot to characterize respectively the raft and non-raft domains in the fractions recovered in our experimental conditions.

Nearly 80% of the membrane proteins were in the flotillin-positive fractions. Whereas the Gadph-positivefractions contained a low amount of cholesterol, free fatty acids and phosphatidylcholine, about 90% of cholesterol and phospholipids were found in the rafts that contained also all plasma membrane-derivedsphingolipids, i.e.ceramides, neutral glycolipids, sphingomyelin and gangliosides. Our results suggest that the plasma membranes of rat hepatocytes are made mostly of domains highly resistant to the action of detergents.

Sphingomyelin and STAT3 in nuclear lipid rafts.

G. Cascianelli, M. Villani, M. Tosti, MP Viola Magni, E. Albi
Department of Clinical and Experimental Medicine, Physiopathology Section, Policlinico Monteluce, 06100, Perugia. ealbi@unipg.it

Lipid rafts are microdomains enriched in cholesterol and glycosphingolipids and are detected mainly in cellular membranes. Because of the high amount of cholesterol and sphingolipids, the fluidity of these regions is lower than the other regions of the membrane. The fatty-acid chains of lipids within the rafts tend to be extended, highly saturated and so more tightly packed, creating domains with higher order, and a new phase calledliquid-ordered phase (lo phase, Zuckermann, 1993).

Many proteins with a high affinity for the lo phase have been found in this well organised phase: GPI- anchored proteins, Src-family kinases and heteromeric G proteins.

The presence of different proteins endows to the rafts with many functions including cholesterol transport, endocytosis, protein trafficking processes and signal transduction (Moffet et al., 2000).

The demonstration of the existence of a nuclear lipid fraction highly enriched in sphingomyelin and cholesterol allowed us to hypothesise the existence of lipid rafts also in the nuclear membrane.

Therefore the aim of the present work was to study the possible presence of lipid rafts in hepatocyte nuclei. The lipid rafts were purified, after Triton X-100 treatment at 4°C, according to Danielsen (1995).

The electron microscopy analysis revealed microdomains with a typical and defined bilayer and a morphology similar to those previously studied in the cellular membrane.

The biochemical analysis showed a higher amount of sphingomyelin than that detected in other nuclear compartments, particularly the nuclear membrane. Likewise, the cholesterol content in nuclear rafts was higher than the cholesterol detected in other nuclear structures, and the ratio cholesterol-sphingomyelin is quite similar to cellular membrane lipid rafts, confirming the typical composition of these structures. Sphingomyelinase activity was also present in nuclear lipid raft.

Protein analysis evidenced many proteins of different molecular weight, with a very evident spot corresponding to a molecular weight of 90 KDa. Since sphingomyelinase is involved in STAT 3 activation, we evaluated the possible presence of this protein by immunoblotting analysis using specific antibodies. The analysis showed positivity for the signal corresponding to 90 KDa.

Thus, this protein is STAT 3, a polypeptide involved in signal transduction, able to modulate the expression of different genes involved in the cell cycle, proliferation and apoptosis. This confirms the important role that rafts can play as a platform for mediators of cell processes, also at a nuclear level. The next step will be the characterisation of the other proteins found to determine all the roles of these microdomains.

Danielsen E.M., Biochemistry 34 (1995), 1596-1605

Moffet S. Brown D.A., and Linder M.E., J. Biol. Chem. 275 (2000), 2191 – 2198

Jørgensen K., J.H. Ipsen, O.G. Mouritsen, and M.J. Zuckermann, Chem Phys. Lipids 65 (1993), 205 – 216

Regulation of cell migration by lipid phosphates and their phosphatases.

D. Brindley
Signal Transduction Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2

ABSTRACT NOT RECEIVED

Modeling and mutagenesis study of the pleckstrin homology domain of ceramide kinase.

Frédéric Bornancin, Markus Jaritz, Piroska Dévay, Siegfried Hoefinger, Philipp Rovina, Andreas Billich, Thomas Baumruker
Novartis Institutes for BioMedical Research, Brunner strasse 59, A-1235 Vienna, Austria

The N-terminus of ceramide kinase (CERK) bears a Pleckstrin Homology (PH) domain. We recently showed that this domain is essential for kinase activity and localization as well as vesicular trafficking of the enzyme1. We now used computational modeling and site-directed mutagenesis to investigate the properties of the CERKPH-domain. In silico protein folding analysis unambiguously confirmed that the N-terminal part of CERK (a.a.8-121) is best fitted to a PH-domain fold. The top hit obtained after threading is mouse Tapp2 N- terminal PH domain, and we therefore modeled CERK PH-domain on the basis of the known 3D-structure of Tapp2. Analysis of the CERK PH-domain model using Poisson-Boltzmann calculations, indicates a strong positive electrostatic potential, in line with the ability of CERK to bind membranes and phosphoinositides. InPH-domains, three variable loops, VL1, VL2 and VL3, interconnecting strands β1-β2, β3-β4 and β6-β7,respectively, provide key charged residues interacting with phosphate groups of phosphoinositides. The VL3 loop of CERK PH-domain appears to be unusually long and to play an important role, as seen from the behavior of recombinant proteins mutated within this region. Furthermore, the profile of phosphoinositide binding to the CERK PH-domain is consistent with the ability of the enzyme to readily associate with various intracellular components, e.g. Golgi complex, vesicles and plasma membrane.

1Carré et al. Biochem. Biophys. Res. Comm. (2004) 324:1215-19.

Sphingomyelin synthase 2 regulates NF-KB activation in hela cells.

Maristella Villani^1,2, Young Choi¹, Maurizio Del Poeta¹, Y. A. Hannun¹, and Chiara Luberto^1
1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425 and ²Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100 Perugia, Italy

Sphingomyelin synthase (SMS) represents an important class of enzymes regulating sphingolipid metabolism. In particular, SMS transfers the phosphorylcholine moiety from phosphatidylcoline onto ceramide forming sphingomyelin and diacylglycerol (DAG). Because of the ability to modulate in opposing directions the levels of ceramide, a negative regulator of cell growth, and DAG, a well-established mitogenic factor, SMS activity has been proposed to play a significant role in the regulation of those processes associated with aberrant cell proliferation. Recently, two sphingomyelin synthases, SMS1 and SMS2 have been identified. Both enzymes showed bidirectional activity when tested in vitro after expression in yeast cells, and when expressed in mammalian cells, SMS1 localized to the Golgi apparatus whereas SMS2 localized to the Golgi and plasma membrane. Our laboratory has previously shown that activation of a plasma membrane associated SMS would lead to nuclear translocation of NF-kB, a transcription factor which promotes proliferation and inflammation. Based on these observations, we investigated the function of SMS2 in mammalian cells and its role in the regulation of NF-kB activation. Treatment of Hela cells with silencing RNA (siRNA) to specifically down regulate SMS2 led to a 40-45% decrease of total SMS activity in vitro. Although no change in the mass level of DAG was found upon SMS2 down-regulation, an increase of ceramide and decrease of sphingomyelin levels were observed, suggesting a basal SMS function for SMS2 in cells. Down-regulation of SMS2 caused inhibition of basal as well as serum- and TNF-induced NF-kB activation as determined by decreased translocation to the nucleus (western blot and immunofluorescence) and DNA-binding activity (Electrophoretic Mobility Shift Assay). Finally, down-regulation of SMS2 significantly inhibited basal and induced expression of a NF-kB target gene, such as cox-2 as determined by real time PCR and luciferase reporter gene assay. All together these results suggest that SMS2 functions as a SMS in Hela cells, and participates in maintaining the molecular machinery that controls proper NF-kB activity.

∗This work was in part supported by COBRE Grant #5NIHP20RR17677 to LM Obeid (project #6 to C. Luberto; mentor, Dr James S. Norris).

Ceramide-1-phosphate: a key metabolite for regulation of cell survival.

Antonio Gómez-Muñoz¹, Jennifer Kong², Kuljit Parhar², Sherry Wang², Patricia Gangoiti¹, Mónica González, Vince Duronio², and Urs P. Steinbrecher^2
1Department of Biochemistry and Molecular Biology, University of the BasqueCountry, Bilbao (Spain), and ²Department of Medicine, University of British Columbia, Vancouver (Canada).

Ceramide-1-phosphate (C1P) is a bioactive sphingolipid that regulates vital cellularprocesses. We first showed that C1P is mitogenic for fibroblasts, and more recently wehave demonstrated that it prevents apoptosis in bone marrow-derived macrophages.

This action of C1P involves direct inhibition of acid sphingomyelinase (A-SMase) andblockade of the caspase 9/caspase 3 pathway. Current work in our laboratory indicatesthat C1P is a potent stimulator of the phosphatidylinositol 3-kinase (PI3-K)/proteinkinase B (PKB, Akt) pathway, which is a major mechanism whereby growth factorspromote cell survival. Pretreatment of the macrophages with the selective PI3-Kinhibitors LY294002 and wortmannin blocked the inhibitory effect of C1P on cellapoptosis. However, C1P did not stimulate mitogen-activated protein kinases (MAPK), and the MAPK kinase inhibitors PD98059 and UO126 did not alter the effect of C1P onmacrophage survival. By looking into downstream effectors of PKB that might beaffected by C1P we were able to show that C1P enhanced the DNA binding activity ofthe transcription factorNF-kB. Interestingly, the selective NF-kB inhibitors, caffeic acidphenylethyl ester and SC- 514, blocked the prosurvival effect of C1P. In addition, apoptotic macrophages showed a marked reduction of Bcl-XL levels, and this decreasewas prevented by C1P. Taken together, these findings suggest that C1P blocksapoptosis, at least in part, by stimulating the PI3-K/PKB/NF-kB pathway andmaintaining the production of antiapoptoticBcl-XL.

We propose a working model for C1P in which inhibition of A-SMase and thesubsequent decrease in ceramide levels would allow cell signaling of C1P through stimulation of the PI3-K/PKB pathway to promote cell survival.

This work was supported by grants MT8630 from CIHR (Canada) and SAF-2002-03184 from Ministerio de Ciencia y Tecnología (Spain).

Relative ceramide and ceramide-1-phosphate levels: a cellular gauge for cell death during development.

Y. León^1,2, T. Homan¹, E.J. de la Rosa³, I. Varela-Nieto¹.
¹Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM. Arturo Duperier 4, 28029 Madrid, Spain. ²Dpto. Biología (Fisiología Animal), UAM. Darwin 2, 28049 Madrid, Spain. ³Centro de Investigaciones Biológicas, CSIC. 28040 Madrid, Spain.

Development involves the dynamic balance of cell division, differentiation and programmed cell death. The ex vivo culture of intact inner ear primordium, the otic vesicle, provides an excellent model system to study the physiological interactions among extrinsic factors during early development.

We have described the opposite actions of nerve growth factor (NGF) and insulin-like growth factor-I (IGF-I)during inner ear ontogenesis. In the otic vesicle, IGF-I is the strongest promoter of inner ear proliferation and morphogenesis, whereas NGF induces programmed cell death by binding to its low affinity p75 neurotrophin receptor (p75NTR). Activation of the p75NTR death domain triggers the generation of ceramide, the activation of caspase-3, the degradation of PARP and finally DNA fragmentation and cell death. Ceramide is generated during cellular stress and apoptosis and has been demonstrated to be a potent inductor of apoptosis within the otic vesicle neuroepithelium (Frago et al., J. Cell Sci. 116, 475. 2003). The exogenous administration of a synthetic short-chain ceramide (C2-cer) induces apoptosis in the otic vesicle. On the contrary,ceramide-1-phosphate (Cer-1-P) is a cytoprotector for otic vesicle explants, acting as a suppressor of cell death upon withdrawal of serum. Addition of Cer-1-P to otic vesicles cultures induces the phosphorylation of Akt and increases PCNA levels. Other lipidic mediators such as phosphatidic acid, sphingosine,sphingosine-1-phosphate or sphingosyl-phosphorylcholine have no appreciable survival actions on this system. NGF induces ceramide generation by activating both de novo synthesis and by increasing the activity of the acid sphingomyelinase. Both NGF- and short-chain ceramide-induced apoptosis are blocked by caspase inhibitors and by IGF-I to different extents. The protective role of IGF-I on NGF- induced cell death was accompanied by a decrease in the intracellular levels of ceramide and by the activation of the Akt kinase pathway. These results suggested that ceramide clearance by means of ceramide kinase activation could be a target of IGF-I in our system. Accordingly, IGF-I actions can be blocked by R59949, an inhibitor of lipid kinases containing a DAGK domain and RT-PCR studies show that ceramide kinase is expressed in the developing chicken otic vesicle. Being its expression up regulated at the stages crucial for cell survival and proliferation. Therefore, the conversion of the pro-apoptotic ceramide to its phosphorylated cytoprotective metabolite ceramide-1-phosphate is one of the pathways underlying the protective action of IGF-I against apoptosis induced by NGF or C2-cer.

Our data suggests that the relative levels of ceramide and ceramide-1-phosphate form part of the panel of the intracellular indicators that regulate cell death decisions during normal development.

Sphingosine-1-phosphate in human glioma cells.

Riboni L., Anelli V., Bassi R., Giussani P., Viani P., Tettamanti G.
Dept. Medical Chemistry, Biochemistry and Biotechnology, LITA-Segrate, University of Milan, Italy

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite, that plays important roles in the regulation of vital cellular processes, acting as both an intracellular messenger and an extracellular ligand for specific transmembrane receptors. As it occurs in many cells, a hallmark of S1P action in glioma cells is the induction of proliferation and protection against apoptosis, implicating S1P in glial transformation. Although information on receptor-mediated functions of S1P is available, little is known on the cell type(s) that may produce extracellular S1P in gliomas. In this study we searched for the possible origin of extracellular S1P in human glioma cells, using T98G, a human glioblastoma cell line, as model. After feeding T98G cells with tritiated sphingosine, S1P was rapidly synthesized, the majority of it undergoing degradation. In these conditions, cell medium analysis revealed the presence of extracellular radioactive S1P, whereas sphingosine kinase activity was undetectable. The extracellular export of S1P was induced by different mitogenic stimuli. In growth-stimulated astrocytes, the administration of N,N-dimethylsphingosine (a sphingosine kinase inhibitor) resulted in a significant reduction of S1P release and cell proliferation. Moreover, the administration of S1P to quiescent T98G cells was followed by the stimulation of cell growth. a rapid activation of ERK1/2, (mediated through a PTX-dependent pathway) and. In addition, we found that bEnd5, a brain-derived endothelial cell line, can release in the extracellular milieu both S1P and sphingosine kinase. The amount of S1P released from bEnd5 was markedly increased after co-culture with T98G cells. Altogether this study supports that S1P acts as an autocrine/paracrine growth factor in T98G glioma cells, and may be implicated in glial tumors.

Sphingosine kinase-1 mediates IGFBP-3 proangiogenic effects in human endothelial cells.

^1,2Riccarda Granata, ³Enrico Lupia, ^1,2Letizia Trovato, ^1,2Fabio Settanni, ^1,2Silvia Destefanis, ^1,2Cristina Barbirato, ^1,2Davide Gallo, ²Giovanni Garbarino, ⁴Riccardo Ghidoni, ^2,5Giovanni Camussi, ²Ezio Ghigo
¹Laboratory of Molecular and Cellular Endocrinology, ²Department of Internal Medicine and ³Clinical Physiopathology, University of Torino, Torino, Italy; ⁴Laboratory of Biochemistry and Molecular Biology, San Paolo Medical School; University of Milano, Milano, Italy; ⁵Research Center for Experimental Medicine (CeRMS), Ospedale S. Giovanni Battista, Torino, Italy.

Angiogenesis, the formation of new vessels from pre-existing vessels is a key step in developmental and pathological events, including wound healing and tumorigenesis. We have recently shown that insulin-likegrowth factor binding protein-(IGFBP)-3, the major carrier of IGF, inhibits human endothelial cell apoptosis induced by serum starvation by decreasing the proapoptotic sphingolipid ceramide. Moreover, IGFBP-3activated sphingosine kinase (SphK), that catalyses the formation of sphingosine-1-phosphate (S1P), a lipid second messenger known to inhibit endothelial cell apoptosis and to induce angiogenesis. IGFBP-3 even increased IGF-I expression, induced IGF-I receptor, Akt and ERK1/2 phosphorylation and enhanced cell motility. On the basis of the above result, we investigated the role of IGFBP-3 in human endothelial cell angiogenic signaling. We show that IGFBP-3 dose-dependently promoted capillary-like structure formation by endothelial cells on growth factor-reduced Matrigel. cDNA array analysis showed that IGFBP-3 enhanced the expression of 8 angiogenic-related genes. The use of other methods (RT-PCR, ELISA and gelatin zymogram analysis) provided further evidence of increased expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP)-2 and -9. Importantly, IGFBP-3 also increased the production ofmembrane-type 1 MMP (MT1-MMP), that plays a key role in MMP-2 activation. Decreased SphK1 expression by small interfering RNA (siRNA), blocked the proangiogenic effects of IGFBP-3 on capillary-like formation and inhibited VEGF and MMPs but not the previously observed IGF-I up-regulation. Finally, IGFBP-3dose-dependently stimulated the growth of neovessels into subcutaneous (s.c.) implants of Matrigel in vivo. We propose IGFBP-3 to be a positive regulator of angiogenesis. Moreover, we suggest that SphK1 mediatesIGFBP-3 proangiogenic effects in human endothelial cells.

Regulation of autophagy by sphingosine -1-phosphate.

Lavieu G¹, Scarlatti F², Sala G², , Levade T³, Ghidoni R², Botti J¹, Codogno P^1
1INSERM U504 Villejuif France; ²University of Milan Italy; ³INSERM U466 Toulouse France

Autophagy is a process by which cytoplasm is delivered to the lysosome and degraded. Autophagy is involved in the control of cell fate by promoting cell survival (under starvation or during cell invasion by bacterial and viral pathogens). Moreover autophagy is associated with non classical cell death under anti- tumoral treatment (as tamoxifen treatment in MCF-7 cell line).

Sphingolipid are equally involved in regulation of cell viability by an equilibrium between ceramide induced cell death and sphingosine-1-phosphate induced cell survival.

We have recently shown that ceramide is a mediator of tamoxifen induced autophagy and cell death in MCF- 7 cells (Scarlatti et al 2004 J. Biol. Chem.).

Here we show that sphigosine-1-phosphate stimulates autophagy by increasing the formation of LC3 positive autophagosomes and the rate of 3-methyladenine sensitive proteolysis without modification of the level of ceramide. Sphingosine kinase 1 (SK1) overexpression recapitulated the stimulation of S1P supplementation on autophagy. This effect was counteracted by the SK inhibitor dimethysphingosine and by expression of a dominant negative form of SK1. Dihydrosphingosine 1-P, an agonist of S1P cell surface receptors, did not stimulate autophagy supporting the notion that autophagy is mediated by the intracellular second messenger activity of S1P. In contrast to ceramide-mediated autophagy, S1P-mediated autophagy is characterized by inhibition of mTOR signaling independently of the Akt/PKB signaling arm. In contrast to ceramide, S1P did not trigger autophagic cell death. Moreover S1P-mediated autophagy has a protective effect toward the induction of apoptosis by nutrient and serum free medium. In conclusion both ceramide and SP1 are able to stimulate autophagy by different pathways with opposite outcomes on cell survival and death.

Sphingosine kinase-1 as a chemotherapy sensor in prostate adenocarcinoma cell and mouse models.

Dimitri Pchejetski¹, Muriel Golzio², Cyril Calvet^1,3, Elisabeth Bonhoure¹, Nicolas Doumerc^1,3, Virginie Garcia¹, Catherine Mazerolles⁴, Pascal Rischmann³, Justin Teissié², Bernard Malavaud³, Olivier Cuvillier^1
1Inserm U466, Institut Louis Bugnard, BP 84225, 31432 Toulouse Cedex 4, France ; ²Institut de Pharmacologie et de Biologie Structurale, CNRS UMR5089, Route de Narbonne, 31077 Toulouse; ³Service d’Urologie et de Transplantation Rénale, Hôpital Rangueil, TSA 50032, 31059 Toulouse Cedex 9; ⁴Service d’Anatomie et Cytologie Pathologiques, Hôpital Rangueil, TSA 50032, 31059 Toulouse Cedex 9, France.

Systemic chemotherapy was considered of modest efficacy in prostate cancer until the recent introduction of taxanes. We took advantage of the known differential impact of camptothecin and docetaxel on human PC-3and LNCaP prostate cancer cells to determine their effect on sphingosine kinase-1 activity and subsequent ceramide/sphingosine 1-phosphate balance, in relation to cell survival. In vitro, docetaxel and camptothecin induced strong inhibition of SphK1 and elevation of ceramide/S1P ratio only in cell lines sensitive to the drugs. Sphingosine kinase-1 overexpression in both cell lines impaired the efficacy of chemotherapy by decreasing ceramide/S1P ratio. Alternatively, silencing sphingosine kinase-1 by RNAi or pharmacological inhibition induced apoptosis coupled with elevation of ceramide and loss of S1P. Differential effect of both chemotherapeutics was confirmed in an orthotopic PC-3/GFP model established in nude mice. Docetaxel induced stronger sphingosine kinase-1 inhibition and ceramide/S1P ratio elevation than camptothecin. This was accompanied by smaller tumor volume and reduced occurrence and number of metastases. Sphingosine kinase-1overexpressing PC-3 cells implanted in animals developed – not only remarkably larger tumors – but also resistance to docetaxel treatment. These results represent the first in vivo demonstration of sphingosinekinase-1 as a sensor to chemoth.

Resveratrol affects the formation of multicellular spheroids of breast cancer cells: involvement of ceramide and adhesion molecules.

Leda Roncoroni¹, Giusy Sala², Floriana Facchetti³, Elena Dogliotti¹, Nicoletta Sacchi⁴, Caterina La Porta³, Ersilia Dolfini¹ & Riccardo Ghidoni^2
1Dept Biology and Genetics and ²Lab Biochemistry and Molecular Biology, San Paolo Medical School, ³Dept of Biomolecular Science and Biotechnology, University of Milan, Italy; 4Dept Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA

Resveratrol, a polyphenol present in grapes and wine, exerts a drastic growth-inhibitory effect on the human breast cancer MDA-MB-231 cell line grown in the 2D-culture. We recently described the mechanistic relationship between growth inhibitory/pro-apoptotic effect of resveratrol and de novo synthesis of ceramide [Scarlatti et al FASEB J (2003) 17,2339-41].

Here we report that ceramide mediates the growth inhibitory effects of resveratrol in MDA-MB-231 cells grown in three-dimensional (3D) cultures. MDA-MB-231 when grown in 3D-cultures can form multicellular tumour spheroids (MCTSs), which revealed an ovoid or spherical shape with well-defined and compact cells, smooth boundaries and regular surface.

First, we observed that treatment of MDA-MB-231 cells with 32 µM resveratrol significantly impaired the formation (number and size) of 3D-MCTSs. This effect correlated with a time-dependent accumulation of endogenous ceramide. In addition, cells disaggregated from the few MCTSs that were able to form MCTSs after resveratrol treatment lost their clonogenic potential.

The levels of expression of E-cadherin, N-cadherin, P-cadherin, PECAM (CD31), P-selectin and VCAM were also analysed by RT-PCR in MDA-MB231 3D-MCTSs. For E-cadherin and VCAM factors no significant expression were detected. In contrast, N-cadherin, P-selectin and P cadherin factors were expressed byMDA-MB231 cells, but 3D conditions (7 and 10 days spheroids) did not modify significantly their level of expression.

The treatment with resveratrol dramatically decreased the level of expression of N-and P-cadherin, while the level of CD31 appeared unmodified in all the growth condition (2D or 3D). Interestingly, in cells grown as spheroids resveratrol was more efficacious than in cells grown on plastic plate. In particular, 32 µM resveratrol was efficacious up to 10 days.

Thus, resveratrol can affect the formation of MCTSs by triggering ceramide-dependent pathways and modifying expression of adhesion molecules.

Cannabinoids induce apoptosis of glioma cells via ceramide- dependent up-regulation of the pro-apoptoticgene p8.

Arkaitz Carracedo, Cedric Malicet^§, Ainara Egia, Cristina Blázquez, Mar Llorente, Raquel Villuendas#, M. A. Piris#, Juan L. Iovanna^§, Manuel Guzmán and Guillermo Velasco
Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, 28040 Madrid Centro Nacional de Investigaciones Oncológicas, 28029 Madrid# – U624 INSERM, Campus de Luminy, Marsella, Francia^§

Cannabinoids – and in particular D9-tetrahydrocannabinol (THC) – have been shown to exert antiproliferative actions both in vitro and in vivo on a wide spectrum of tumour cells. In the case of gliomas and leukaemias, this effect is – at least partially – due to the ability of these compounds to induce apoptosis of these cells via stimulation of ceramide synthesis de novo. In order to understand the molecular mechanisms underlying thispro-apoptotic effect, we have studied the gene expression profile of two sub-clones of the rat glioma cell line C6 that exhibit different sensitivity to the treatment with cannabinoids.

Our results show that one of the genes that are differentially expressed in the cannabinoid-treated sensitive cells is the stress-regulated gene p8. By using p8 siRNA and a retroviral vector to over-express this protein, we further confirmed that expression of this gene was necessary for the pro-apoptotic effect of cannabinoids to be observed in glioma cells. Interestingly, pharmacological inhibition of ceramide synthesis de novo prevented not only cannabinoid-induced apoptosis but also p8 up-regulation. Moreover, abrogation of this pathway also prevented up-regulation of several downstream targets of p8 that seem to participate in cannabinoid-inducedapoptosis.

In summary, our results suggest that treatment with cannabinoids leads to apoptosis of glioma cells via aceramide-dependent up-regulation of the pro-apoptotic gene p8.

Effect of vitamin D3 on embryonic hippocampal cells via neutral sphingomyelinase.

F Marini¹, Bartoccini E¹, Viola Magni M¹, Garcia-Gil M² and Albi E¹,
¹Department of Clinical and Experimental Medicine, Physiopathology Section, Policlinico Monteluce, 06100, Perugia. – ²Dept. of Physiology and Biochemistry, University of Pisa

Over the last ten years abundant evidence has emerged for new roles of vitamin D3. This molecule is known for its activity in calcium preservation and phosphorous homeostasis but it is also involved as a defensive agent against multiple sclerosis and in the prevention of encephalomyelitis progression, suggesting an immunosuppressive action (1). Recently, it has been demonstrated that Vitamin D3 induces erythroleucemic cell differentiation via sphingomyelinase (SMase) and prevents serum starvation apoptosis (2). Since in HN9.10e embryonic hippocampal cell serum deprivation induces apoptosis first increasing the nuclear SMase activity and after cellular SMase activity (3), the aim of the present work was to establish if vitamin D3 could have a role in slowing down the cell cycle inducing differentiation and preventing serum deprivation induced apoptosis via sphingomyelin (SM) metabolism.

HN9.10e cells were synchronized by serum free DMEM for 24h and then were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 2.5 µg/ml amphotericin B (fungizone). In experimental cells 400 nM vitamin D3 was added. Cells were maintained at 37 °C in a 5% CO2 humid atmosphere. Cell cycle was studied using 3H- thymidine whereas the SM metabolism was studied incubating the cells with 3H palmitic acid.

The results show that, after synchronization, the cells present two peaks of 3H-thymidine incorporation at 36 and 60 hrs respectively, corresponding to the first and second mitotic wave. Vitamin D3 does not influence behavior in time but reduces the first peak by 32% and the second by 47%, suggesting a reduction of the cell number entering in the S phase. At 48h hrs of 3H palmitic acid incorporation, the saturation conditions were reached in SM and ceramide. After this time, in the control sample the radioactivity was 45 nmol/mg protein and 35 nmol/mg protein for SM and ceramide respectively and the values did not change up to 120 min. The vitamin D3 determined a 6.6 fold increase in SM at 120 min and may influence the reduction of the S phase of the cell cycle and induce differentiation. Serum deprivation did not change the SM levels, as previously reported(3) but the vitamin D3 in these cells increased the SM levels after 30 min of treatment. The early increase of SM could play a role in preventing serum deprivation induced apoptosis.

1- A.J. Brown, A. Dusso and . Slatopolsky. Am J Physiol Renal Physiol, (1999) 277: F157-175
2- Witasp E, Gustafsson AC, Cotgreave I, Lind M, Fadeel B. Biochem Biophys Res Commun. (2005) 330:891-7.
3- E.Albi, S. Cataldi, E. Bartoccini, M. Viola Magni, F. Marini, F. Mazzoni, G. Rainaldi, M. Evangelista, M. Garcia-Gil2005 J. Cell Physiol, in press.

Sphingomyelin derivatives affect the progress of denervation of rat soleus muscle.

M.Zanin¹, E. Germinario¹, R.A. Sabbadini², R, Betto³ and D. Danieli^1
1Department of Human Anatomy and Physiology, University of Padova, Italy; ²Department of Biology, San Diego State University, USA, 3C.N.R. Neuroscience Institute, Muscle Biology and Physiopathology Unit, Padova, Italy.Via Marzolo 3, 35131 Padova. Tel. 049 8275305, Fax. 049 8275301

Present work is aimed at studying the effects of bioactive sphingomyelin derivatives on the progress of denervation of slow-twitch skeletal muscle. The study is based on the fact that sphingomyelin derivatives are elevated in serum so that we hypothesize they may exert trophic functions on muscles. Denervation was bilaterally performed cutting the sciatic nerve at the level of trochanter in adult rats. Sphingosine (SPH), sphingosine 1-phosphate (S1P) or sphingosylphosphorylcholine (SPC) were continuously released over soleus muscle by a mini-osmotic pump implanted subcutaneously in the scapular region and connected to the muscle through a catheter. Effects of individual sphingomyelin derivatives were evaluated on the fibre cross sectional area, myosin heavy chains composition (MHC), TUNEL-positive nuclei, and expression of MRFs. In general, either of the three lipids decrease the development of atrophy, with SPH and SPC being the most effective. However, the action of the lipids appears to be to some extent distinctive. The protective action of SPC has an early onset and is transient, while that of SPH is delayed. SPH slows down the slow- to-fast transformation of MHC isoforms normally occurring in denervation, while S1P and SPC do not seem to influence this process. SPH, S1P and SPC modulate the expression of MyoD and myogenin, suggesting that the three lipids influence satellite cells.

In general, SPH and S1P/SPC exert distinctive and opposite actions. SPH inhibits cell proliferation and promote apoptosis, while the extracellular action of S1P/SPC stimulates cell growth and protect from apoptosis. In contrast, present results show that the three lipids produce common effects, even though distinctive, on the progress of soleus muscle denervation. We speculate that the expected negative effect of SPH, that is the worsening of atrophy, is abolished by the presence at muscle fibre surface of SPH kinase, which convert SPH in S1P. Preliminary data show that in the presence of SPH kinase inhibitor, DMS, the SPH positive effect is lost. Future work will further investigate this hypothesis and the molecular mechanisms at the base of the distinctive actions of sphingolipids on skeletal muscle.

Funded by PRIN 2003, NIH, AHA and CNR.

Control of bioactive sphingolipid levels by retinoic acid receptors in breast cancer cells.

Giulia Somenzi¹, Giusy Sala², MingQiang Ren¹, Manuela Amelotti¹, Riccardo Ghidoni², Nicoletta Sacchi^1
1Dept. Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, 14263, USA – ²Lab. Biochemistry & Mol Biology, San Paolo University Hospital, Medical School, University of Milan, Milan, Italy

All-trans-retinoic acid (RA) is a bioactive natural derivative of Vitamin A. RA action is mediated by nuclear RA receptors, RARs. More than one RA-receptor is involved in mediating RA-signal. RA binds RARα, which in turn regulates another RA receptor, RAR β2, with potent tumor suppressor activity. RA-resistant cancer cells often lack both RARα and RARβ2 expression. Lack of both RARs has been traced to epigenetic silencing, due to aberrant DNA methylation and histone modifications of the RARs’ chromatin. Breast cancer cells lacking both RARα and RARβ2 are unable to respond to the growth inhibitory, differentiating and apoptotic effects of RA. Biologically active sphingolipids like ceramide and sphingosine-1-phosphate (S1P) are pivotal regulators of these fundamental cellular processes. We found that sphingolipid metabolism is different in RA-resistantbreast cancer cells lacking both RARα and RARβ2 and RA-sensitive cells with intact RARα and RARβ2. We hypothesized that RARs control sphingolipid metabolism. Here we show that as a consequence of functional RARα disruption, breast cancer cells become unable of a) re-inducing RARβ2 in response to RA, b) accumulating ceramide via neutral sphingomyelinase and c) reducing the level of sphingosine kinase transcription and activity. Moreover, cells instead of being growth-inhibited are growth- promoted by RA. However, cells retain the ability of accumulating endogenous ceramide and undergoing apoptosis in response to the synthetic retinoid fenretinide, which action is RAR-independent.

The overall data lead to the conclusion that RARs control distinct sphingolipid metabolic pathways.

Activation of APP1 transcription by ipc1-dag pathway is regulated by ATF2 in Cryptococcus neoformans.

Roberta Iatta^1,2, Lydia Mare², Chiara Luberto², Maria Teresa Montagna¹ & Maurizio Del Poeta^2
1Dipartimento di Medicina Interna e Medicina Pubblica Sezione Igiene, Università degli Studi di Bari, Bari, Italia – ²Department of Biochemistry and Molecular Biology Medical University of South Carolina, Charleston, South Carolina, USA

Inositol-phosphoryl ceramide synthase 1 (Ipc1) is a fungal specific enzyme that regulates the level of two bioactive molecules, phytoceramide and diacylglycerol (DAG) (1). In previous studies, we demonstrated that Ipc1 regulates the expression of antiphagocytic protein 1 (App1), a new factor involved in the pathogenicity of Cryptococcus neoformans (2). Here, we investigated the molecular mechanism by which Ipc1 regulates App1. To this end, the APP1 promoter was fused to the firefly luciferase gene in C. neoformans wild-type and in GAL7::IPC1 mutant, in which Ipc1 expression can be modulated. We found that luciferase activity is indeed regulated by modulation of Ipc1, suggesting that the lipid(s) regulated by Ipc1 may trigger APP1 transcription. Next, we investigated the role of DAGs and other sphingolipids in the activation of APP1 promoter and we found that treatment with 1,2-dioctanoyl-glycerol increases luciferase activity whereas treatment with phytosphingosine or ceramides did not affect luciferase activity. Importantly, deletion of ATF2 consensus sequence in the APP1 promoter or deletion of ATF2 gene from the genome abolishes luciferase activation by Ipc1 or DAG, suggesting that Atf2 transcription factor is sufficient and necessary for regulation of APP1 gene transcription. Since App1 is found in sera of AIDS patients affected by cryptococcosis, these studies may have important implications in understanding mechanisms of pathogenicity of C. neoformans.

References:
Luberto C, Toffaletti D, Wills E, Tucker S, Casadevall A, Perfect J, Hannun Y, Del Poeta M. Roles for inositol-phosphorylceramide synthase 1 (IPC) in pathogenesis of C. neoformans . Gene & Development 2001; 15: 201-212.
Luberto C, Martinez-Marino B, Taraskiewicz D, Bolanos B, Chitano P, Toffaletti D, Cox G, Perfect J, Hannun Y, Balish E, Del Poeta M. Identification of App1 as a regulator of phagocytosis and virulence of Cryptococcus neoformans. J. Clin. Invest. 2003; 112: 1080-1094.

The presence of the inositolphosphoceramide synthase AUR1p is critical for the survival of lac1 lag1 yeast mutants lacking the acyl-coa-dependent ceramide synthase.

Isabelle Guillas¹, Vanessa Cerontola, S. Michal Jazwinski² and Andreas Conzelmann
Department of Medicine, University of Fribourg, Switzerland and ²Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, USA.

The lag1∆ lac1∆ double mutants lack acyl-CoA-dependent ceramide synthase and are either very sick or nonviable. Growth of lag1∆lac1∆ cells can be greatly improved by overexpression of Ypc1p or Ydc1p, two homologous alkaline ceramide hydrolases that also catalyze the reverse reaction, i.e. the condensation of free fatty acids with phyto- or dihydrosphingosine (Schorling et al., 2001; Mao et al., 2000a; Mao et al., 2000b). Overexpression of Ypc1p restores viability and growth YPK9 lag1∆ lac1∆ and allows for the synthesis of large amounts of structurally normal inositolphosphorylceramides containing fatty acids with no less than 26 C atoms. Overexpression of Ydc1p also restores viability and slow growth to lag1∆ lac1∆ cells but allows for synthesis of only small amounts of a single C26-containg non-hydroxylated inositolphosphorylceramide. The lag1∆ lac1∆ cells making only limited amounts of ceramides can during a few cell devisions in the presence of low concentrations of Aureobasidine A, a specific inhibitor of inositolphosphorylceramide sybnthase. The drug is taken up as it efficiently blocks residual inositolphosphorylceramide biosynthesis in those cells. Ultimately however, cells stop growing. Also, the AUR1 gene cannot be deleted in these lag1∆ lac1∆ cells overexpressing Ydc1p. The results indicate that the inositolphosphorylceramide synthase AUR1 remains essential in the absence of acyl-CoAdependent ceramides synthase. AUR1 may allow cells to make very small amounts of essential inositolphosphorylceramides or may serve another, unrelated, but essential function.

Involvement of bioactive sphingolipids in the postoperative intestinal trauma.

Mihaela Dragusin, Roland Broere, Sven Nätzker, Sven Wehner^§, Jörg C.Kalff§, Nicolas Schwarz^§, Elaine Wang*, Cameron Sullards*, Alfred H. Merrill Jr.*, Gerhild van Echten-Deckert
Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany ^§ Klinik und Poliklinik für Allgemein-, Viszeral-, Thorax-, und Gefäßchirurgie, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany *School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230,USA

There is growing evidence that inflammatory processes are accompanied by increasing levels of bioactive sphingolipids including ceramide and sphingosin-1-phosphate (S1P). Recently, a novel bioactive sphingolipid,ceramide-1-phosphate (Cer1P) has emerged as an inducer of inflammatory responses by activation of PLA2. However, no information concerning the role of these sphingolipids in inflammatory events during the postoperative intestinal trauma is yet available.

In the present study we analysed the levels of bioactive sphingolipids of mucosa and muscularis from small intestine of i) a standardized surgical rat model of intestinal manipulation, as well as of ii) lipopolysaccharide (LPS) treated rats. In addition, we have used cultured muscularis smooth muscle cells in order to investigate the bioactive sphingolipid downstream targets involved in the inflammatory cascade.

Using thin layer chromatography we detected a slight increase (up to 2 fold) of ceramide levels at 6 h and 3 h after mechanical manipulation in intestinal mucosa and muscularis, respectively, compared to sham controls. Concerning LPS treatment the increase of ceramide was less pronounced and occurred only after 24 h in both intestinal mucosa and muscularis, when compared with sham controls.

Results obtained from ESI tandem mass spectrometry (MS) measurements indicate that not ceramide but rather its metabolic products, Cer1P and S1P are increased in the intestinal muscularis, the mainly affected tissue during postoperative ileus.

Experiments performed in primary cultured muscularis smooth muscle cells indicate that both, ceramide and S1P induce an activation of cox2, a key player in inflammatory processes. Accordingly, increased levels of prostaglandins could be detected. Furthermore, using DNA chip technology, up-regulation of pro- inflammatory cytokines including IL-1 was observed.

Our latest results obtained in primary cultured intestinal smooth muscle cells concerning sphingolipid mediatedpro-inflammatory signalling cascades will be presented.

Psychotic forms of late manifesting adult metachromatic leukodystrophies : phenotype / genotype relationships.

Nicole Baumann, Benoit Colsch, Mireille Lefevre and Jean-Claude Turpin
Laboratory of Neurochimie Unité Mixte de Recherche INSERM U711-UPMC , Hopital de Salpetriere, Paris, France

Metachromatic leukodystrophy (MLD) is due to an abnormal catabolism of sulfogalactosylceramides (sulfatides)and other sulfogalactolipids (seminolipid) because of a deficient cerebroside sulfate sulfatase (arylsulfatase A i.e. ASA).Diagnosis is based on sulfatiduria and ASA determination.

In childhood this disease is multisystemic and its symptomatology and natural history is well known. Surprisingly, this genetic disease can manifest for the first time during adolescence and/or adulthood. In this case, the clinical presentation and evolutivity are very different.

Its presentation may be that of a degenerative disease of the central nervous system with mainly spastic manifestations or a spino-cerebellar ataxia, or that of a psychosis.

To try to understand this psychotic phenomena, we decided to do a complete study reviewing cases already published for which we could have more information, and also unpublished cases from clinics and laboratories including our own. Interestingly several patients were institutionalized in psychiatry departments and considered as schizophrenia. From the survey of the literature and our own experience there seems to be in opposite to infantile forms, a phenotype-genotype relationship in the adult . 10 psychotic adult cases and 3 psychotic juvenile cases responded to the classical criteria of Metachromatic Leukodystrophy, with 5- 10 % normal value, and sulfatiduria. There were common psychiatric pattern according to criteria of the DSM4 for Schizophrenia: delusions, hallucinations,disorganized behavior, affective flattening, social occupational dysfunction. This was associated to cognitive deficiency. Many years could occur till neurological manifestations became evident. Molecular studies were performed both by PCR restriction and sequencing. Motor forms involve the major adult mutation P426L in a homozygote form in contrast to psychocognitive forms which involved as a compound heterozygote a specific I179S mutation of ASA, the second mutation being mainly of the infantile type The I179S mutation was present as a compound heterozygote in the 13 psychiatric cases we studied. Possibly other mutations are also involved in psychiatric cases but I179S is the major one and there is definitely a phenotype/genotype correlation.

Human sialidases: molecular biology, biochemistry and physiopathological implications.

OVERVIEW LECTURE
Guido Tettamanti
Department of Medical Chemistry, Biochemistry and Biotechnology, The Medical School, University of Milan, Via F.lli Cervi 93, 20090 Segrate, Milan, Italy

Sialidases are expressed in viruses, bacteria, protozoa and vertebrates. Viral and bacterial sialidases display about 35% homology in their primary structure, and share some specific motifs: six four-stranded antiparallelβ-sheets arranged as the blades of a propeller; a F(Y)RIP motif located toward the aminoterminal region, a repetitive motif SXDXGXXT/W in the active site region; and some Asp-boxes. In mammalian (including human) organs and tissues, sialidases display different subcellular locations (lysosomes, plasma membrane, cytosol and, possibly, other locations) and substrate specificities. The lysosomal, cytosolic and plasma membrane sialidases, also from the same animal, have different protein structure, antigenic properties and cDNAs.

The following human sialidases have been cloned so far: the lysosomal sialidase NEU1 coded by NEU1 gene mapped in 6p21, the cytosoluble sialidase NEU2 coded by NEU2 gene mapped in 2q37, the plasma membrane bound sialidase NEU3 coded by NEU3 gene mapped in 11q13, and the newly discovered sialidase NEU4 coded by NEU4 gene mapped in 2q37. The corresponding recombinant sialidases constitute powerful tools for studying the structure-function relationships of these enzymes. As an example, recombinant human cytosolic sialidase (Hs NEU2), expressed in E. Coli, was purified to homogeneity and its crystal structure defined. The presence of the canonical six blades β-propeller structure, F (Y)RIP motifs and three Asp- boxes, typical of all studied sialidases, was confirmed. In addition, with the use of the inhibitor DANA (2,3–dehydro–3–deoxy–N–acetylneuraminic acid) the dynamic nature of substrate recognition was assessed. Recombinant Hs NEU2 was proven to differentially recognise: (a) the type of sialosyl linkage (the α 2-3sialosyl linkage to galactose is preferred); (b) the aglycone part of the substrate (gangliosides act as the best substrates); and (c) the supra-molecular organization (monomer, micelle, vesicle) of the ganglioside substrate. The latter ability might be relevant in sialidase interactions with gangliosides under physiological conditions.

Some plasma membrane bound sialidases appear to be attached to the membrane via a glycan- phosphoinositide (GPI) anchor, possibly indicating their location in specialized plasma membrane “domains” (lipid rafts). Surface sialidase NEU3 is able to affect sialoglycolipids exposed on the surface of vicinal cells and was shown to be directly involved in neural and myoblast differentiation. In the latter case the block of sialidase expression led to inhibition of the differentiation toward myocytes. Sialidases appear also to be involved in human erythrocytes maturation and ageing under physiological and pathological conditions. Very recent results point to the implication of sialidase NEU3, and its hydrolysis products gangliosides GM1 and GM2, in the insulin post-receptor signaling events, and of sialidases NEU2 and NEU3 in tumor growth. The genetic lack of sialidase NEU1 is responsible of a very severe lysosomal disease.

Resveratrol sensitization of DU145 prostate cancer cells to ionizing radiation is associated to endogenous ceramide increase.

Scarlatti F., Maioli C.^$, Sala G., Maffei R., Milani F.^$, and Ghidoni R.
Lab Biochem & Molecular Biology – San Paolo University Hospital, Medical School,
^$ Institute of Radiological Sciences, University of Milan, Milan

Radiotherapy is an established therapeutic modality for prostate cancer. Since it is well known that the restriction of radiotherapy is its severe toxicity on normal cells at high dose and minimal effect at low dose. The search for biological compounds able to increase the sensitivity of tumors cells to radiation may improve the efficacy of therapy. Resveratrol, a natural antioxidant, was shown to inhibit carcinogenesis in animal models [1], and to block the process of tumor initiation and progression [2]. The purpose of this study was to examine whether resveratrol can sensitize DU145, an androgen-independent human prostate cancer cell line, to ionizing radiation (IR).

We observed that, although DU145 cells are relatively sensitive to IR-induced cell death, pretreatment with resveratrol significantly enhances tumor cell death induced by IR alone. Resveratrol acts synergistically with IR to inhibit colony formation of DU145 cells in vitro. Moreover resveratrol potentiates IR-induced ceramide accumulation in DU145 cells. We supposed that the clonogenic survival of DU145 cells after combined treatment of resveratrol with IR is associated to the generation of ceramide. The synergistic effect at biologically relevant doses of radiation (0.5-2 Gy) suggests that the combination of resveratrol with IR allows reduction of radiation doses and potentially reduce treatment-related morbidity.

1.Yang CS. et al. Inhibition of carcinogenesis by dietary polyphenolic compounds. (2001), Annu. Rev. Nutr. 21:381-406.
2.Jang M. et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. (1997)Science 275: 218-220.

Effect of dexamethasone on cellular and nuclear sphingomyelin- synthase.

Tringali A. R., Tringali S. , Viola Magni M.P., Albi E.
Department of Clinical and Experimental Medicine, Physiopathology Section, Policlinico Monteluce, 06100, Perugia.

It has been determined that glucocorticoids are essential for cell growth in culture acting upon receptors, enzyme activity and membrane transport (1). On the other hand, these molecules induced growth inhibition and lysis in certain sensitive leukemic cells (2) and apoptosis in mature, activated, peripheral T lymphocytes

(3). The induction of apoptosis could be one of the key mechanisms mediating the therapeutic effect of glucocorticoids in the treatment of leukemias, lymphomas and various autoimmune disorders.

The biological effects of dexamethasone were generally thought to be mediated by an intracellular protein receptor which transduces the hormone signal to the nucleus and participates directly in gene regulation (4). Recently, we have demonstrated that dexamethasone inhibited the growth of lymphoblastic lymphoma cells(SUP-T1) by stimulating the cellular sphingomyelinase (SMase) activity and inhibiting the nuclear SMase activity after two hours of treatment (data not shown). This effect induced an increase in cellular ceramide and a reduction in the nuclear ceramide pool enriching sphingomyelin (SM) in the nucleus. Since the SM- synthase activity was present in the nuclei, the aim of this work was to establish the possible effects of dexamethasone on cellular and nuclear SM-synthase activity in tumor cells. To this end, the lymphoma cells (SUP-T1) were seeded in RPMI 1640 in the presence of 10% fetal bovine serum, at the concentration of 100.000/ml and cultivated up to 6 days in a 5% CO2 humid atmosphere. Two 5% lots were prepared: one control and one cultivated in the presence of dexamethasone (500 µg/ml). The SM-synthase activity was assayed in the homogenate and nuclei by using 3H-phosphatidylcholine as substrate. The results show that in the homogenate the SM-synthase activity is 10 pmol/mg protein/min at 0 min, increases of 80 times after 30 min, then decreases slowly until 2 hours and after remains constant for all considered times. In the nuclei,SM-synthase activity is inhibited by 50% compared to the control after 30 min and then increases progressively up to 2 hours. In conclusion the nuclear SM metabolism is independent from that of in the whole cell and the SM-synthase activity contributes to the SM enrichment pool after two hours of dexamethasone treatment. Since it has been postulated that SM acts on chromatin structure, it is possible that dexamethasone induces apoptosis by enriching the nuclear SM pool and favouring chromatin destabilization.

(1)Nelson DH, Murray DK., Proc. Natl. Acad. Sci. USA. 1982 Nov. 79: 6690-6692.
(2)Yuh YS, Thompson EB., J Biol Chem. 1989 Jun 25; 264(18): 10904-10.
(3)Galili U, J. Steroid Biochem. 1983 ; 19: 483-490.
(4)McEwan IJ, Wright APH, Gustafsson JÅ, BioEssays 1997 ; 19: 153-160

Role of sphingosine kinase 1 in the regulation of proliferation and autophagy in myeloid leukemia cells.

Ricci C.^1,2, Sala G.¹, Onida F.³, Soligo D.³ and Ghidoni R.^1
1Lab Biochem & Molecular Biology – San Paolo University Hospital, Medical School, University of Milan, ²Fondazione Matarelli, Milan, ³IRCCS Ospedale Maggiore and University of Milan

Sphingosine Kinase 1 (SK1) is a key enzyme of the sphingolipid metabolism. By phosphorylating sphingosine (Sph), it induces intracellular accumulation of sphingosine 1 phosphate (S1P). As a second messenger, S1P stimulates proliferation and survival and protects cells against ceramide-induced apoptosis [1]. Although oncogenic activity of SK1 has been demonstrated in solid tumors [2], its role in the proliferation of leukemia cells remains unclear. In particular, we aimed to investigate SK1 involvement in cell growth regulation of chronic myeloid leukemia (CML), an hematopoietic disorder characterized by the malignant expansion of bone marrow pluripotent stem cells. The fusion protein Bcr/Abl, through its constitutive tyrosine kinase activity, is the pathogenetic event that causes CML and thus specific inhibitors have been developed. Among those, Imatinib Mesylate (IM) represents a successful strategy in CML treatment. However, most patients who are treated in advanced stages of the disease relapse after an initial response to IM. Therefore, the existence of a functional link between Bcr/Abl and SK1 would provide alternative targets to develop new strategies to overcome acquired resistance to IM in CML.

Recently, the ERK1/2 and PI3K/AKT pathways were shown to be activated by SK1 overexpression [3]. Those molecules are involved also in the negative control of macroautophagy [4], a constitutive cellular mechanism for the turnover of organelles and long-lived proteins. According to the activating signal, increased autophagy can lead to either cell survival or death. First evidences showed a decrease of autophagic activity in tumor cells, but its exact role in oncogenic transformation needs to be clarified. Autophagosome formation requires the recruitment of Beclin-1, which is the product of a tumor-suppressor gene frequently deleted in human cancer, and LC3, marker of autophagic vacuoles. We aim to investigate the contribution of autophagy to CML proliferation and to define whether there is any relationship between this process and SK1.

As an in vitro model, we used a panel of CML cell lines (KU-812, K562, RWLeu4 and AR230) and evaluated the basal expression level of SK1, Beclin 1 and LC3 by semi-quantitative RT-PCR. Afterwards, the effect of increasing doses (0, 1, 5 and 10 µM) of a novel SK1 inhibitor was determined at different time points (0, 24, 48 and 72 hrs) on cell proliferation and viability. Preliminary results showed that pharmacological inhibition of SK1 caused a decrease of the percentage of viable cells, suggesting that this kinase may play a role in the growth of leukemia cells.

After establishing the IC50 of SK inhibitor for each cell line, the correlations between SK1 and Bcr/Abl will be investigated by treating cells with both SK1 inhibitor and IM. In particular, possible changes in the expression of mRNA and protein together with the kinase activity will be evaluated. Under the same conditions, we will also explore whether autophagy is consequently modulated.

References
1.Cuvillier O et al, Nature, 381: 800-3, 1996
2.French KJ et al, Cancer Res, 63: 5962-9, 2003
3.Lescolan E et al, Blood (published online), 2005
4.Arico S et al, J Biol Chem, 276 (38): 35243-6, 2001

Different ceramide-mediated cell responses to drugs in prostate cancer cells.

Roberta Maffei, Giusy Sala & Riccardo Ghidoni
Lab Biochem & Molecular Biology – San Paolo University Hospital, Medical School, University of Milan

The sphingolipid ceramide is an important bioactive lipid that regulates diverse signaling pathways involving apoptosis, cell senescence, cell cycle and differentiation. It can be generated through different pathways. The various biosynthetic pathways seem to reflect the distinct cellular compartments where ceramide is localized, and to lead to different cellular responses.

Our purpose is to correlate ceramide increase with i) its way of generation, and ii) the biological cell outcome. In this perspective we assayed a panel of different drugs to induce endogenous ceramide increase inandrogen-sensitive prostate cancer cell line (LNCaP).

Celecoxib, CoCl2, doxorubicin, etoposide, fenretinide (4-HPR), resveratrol and tamoxifen have been used. Treated LNCaP have been evaluated for cell growth, induction of apoptosis and ceramide accumulation.

All drugs, with the exception of tamoxifen, decreased viability. Growth curves showed a dose-dependentinhibition of proliferation for each treatment.

With celecoxib, CoCl2, etoposide, 4-HPR and resveratrol we observed a small increase in intracellular ceramide, which is dose-dependent only with 4-HPR.

Ceramide increase is not followed by PARP cleavage, with the exception of 4-HPR, and DNA laddering formation, in a wide concentration range.

These preliminary results suggest that i) the LNCaP biological response to different drugs is not significantlyceramide-dependent, ii) only 4-HPR unequivocally induces apoptosis and this is associated with ceramide increase. The LNCaP genotype (i.e. this cell line exhibits acid ceramidase overexpression [1]) may explain the modest correlation between ceramide accumulation and cell response.

Reference:
[1] Seelan RS, Qian C, Yokomizo A, Bostwick DG, Smith DI, Liu W. “Human acid ceramidase is overexpressed but not mutated in prostate cancer.” Genes Chromosomes Cancer. 2000;29:137-46.

Anti-sphingomyelin antibodies in oncologic patients.

Pugliese L., Bernardini I., Viola Magni MP., Albi E.
Department of Clinical and Experimental Medicine, Physiopathology Section, Policlinico Monteluce, 06100, Perugia.

Antiphospholipid (aPL) antibodies are a heterogeneous group of immunoglobulins1 that exhibit a broad range of target specificities and affinities, all recognizing various combinations of phospholipids, phospholipid-bindingproteins or both and phospholipid- protein complexes. It is known that the high values of Antiphospholipid (aPL) antibodies are indices of Antiphospholipid Syndrome 2 characterised by vascular thrombosis, recurrent pregnancy loss and thrombocytopenia. Recently, the aPL antibody positivity was observed in patients with leukaemia, lymphoma and monoclonal gammopathy3-5.

The aim of this work was to study the possibility of the aPL antibody positivity in patients with solid cancers in which other classic tumour markers were positive.

First, according to the immunoassay currently performed, we used the ELISA Test (ALFA WASSERMANN,Cardiolipina-aK Combi test). Since anticardiolipin antibodies are believed to be polyspecific antibodies thatcross-react with all the anionic phospholipids6, we also proposed the use of phatidylinositol and sphingomyelin as antigens.

Fifty six patients entered the study (35 F; 21 M) average age 62 yrs (range 30-92) with high tumour markers levels (CEA; CA 19.9; Ca 125; CA 15.3). None of the patients had SLE or other conditions known to be associated with aPL antibodies such as chronic infections, recent acute bacterial or viral infections. Patients with inherited causes of venous hypercoagulability (protein C/S, antithrobin III deficiencies) were excluded. As control, 48 healthy donor blood was used. Serum samples were stored at –20 ° C until used.

The results showed that 28 of 56 patients (50.0%) were aPL positive and 25/28 (89.0%), 45% of all patients, had moderate or high antiphospholipid antibody levels. In particular we noticed a positivity for aPL antibodies in patients with high levels of CA 19.09 (6/7 corresponding to 85,7%) or CEA plus CA19.09 (8/10 corresponding to 80%). Nevertheless we also detected a strong association between aSM antibodies and these markers compared to aCL and aPI antibodies. In fact, the results showed that in patients with high level of CEA plus Ca19.09, the IgG positivity was 3/10 (30%) for the CL and PI and 6/10 (60%) for the SM and the IgM positivity were 1/10 (10%) for the CL and PI and 3/10 (30%) for the SM. In patients with Ca19.9 we noticed an IgG positivity for the CL 3/7 (42,8%), for the PI 4/7 (57,1%) and for the SM 3/7 (42,8%), the IgM positivity resulted 0/7 for the CL and PI and 3/7 (42,8%) for the SM.

1.Hughes GRV BMJ 1983; 287: 1088; 2. Jerrold S. Levine M.D., Ware Branch M.D and Joyce Rauch PH.D NEJM, 2002, Vol.346, No.10; 3.Soltez P.,Szekanec Z., Vegh J., Lakos G., Toth L., Szakall S., Veres K., Szegedi G. Haematologia 2000; 30 (4):303-11; 4. Asherson RA, Khamashta MA, Ordi-Ros J, et al. Medicine (Baltimore)1989;68:366-74. 5. Vianna JL, Khamashta MA, Ordi-Ros J, et al. Am J Med 1994;96:3-9.Galli M, Comfurius P, Maassen C, et al. Lancet 1990;335:1544-7.

Effect of energetic stress and the ceramide analogue C6-ceramide on thyroid oncocytoma cell lines.

A.M.Porcelli, A.Ghelli, L.Iommarini, M. Hoque, C. Zanna, V.Carelli*, G. Romeo^§ and M.Rugolo
Dip. di Biologia Evol. Sperim.; *Dip. di Scienze Neurologiche and ^§Dip. di Medicina Interna, Cardioangiologia, Epatologia, Università di Bologna

Thyroid oncocytomas or Hürthle cell tumours (HCT) are characterized by a specific cytoplasmic eosinophilia. This reflects the abundance of mitochondria, which leads to the typical oxyphilic phenotype. Previous biochemical studies have suggested that a mitochondrial dysfunction might underlie HCT (1, 2). Recently, a high frequency of mtDNA mutations in complex I and IV genes has been shown to be involved in thyroid tumours (3, 4), suggesting that a coupling defect in oxidative phosphorylation might be a cause of compensatory mitochondrial hyperplasia in these tumours (5, 6). In order to study the bioenergetic efficiency of mitochondria in HCT we have determined cell growth in a medium where glucose is substituted by galactose. Under these conditions, the rate of glycolysis is greatly reduced and cells are forced to rely only on mitochondria for ATP production. Experiments have been carried out with the XTC-1 cell line (thyroid follicular oxyphilic cells) and the TPC-1 cell line (thyroid papillary carcinoma with no oxyphilic feature). In galactose medium, the TPC-1 cells were able to grow, albeit at a slower rate than in glucose medium. XTC-1 cells on the other hand exhibited a significant decrease in both cell viability and ATP levels. These results suggest that a severe energetic impairment exists in XTC-1 cells, which is likely to be responsible for the compensatory mitochondrial proliferation.

In contrast to the cell death observed in galactose medium, XTC-1 cells were resistant to the toxic effect of the ceramide analogue C6-ceramide (20µM). The ATP content of these cells was maintained after treatment with the ceramide analogue, whereas the ATP level of TPC-1 cells was remarkably reduced. However, both cell lines appeared to be stressed, in particular the mitochondrial network, evaluated after loading with the specific dye Mitotracker Red, was found to be dramatically fragmented and swollen. We observed that Lysotracker Red, which specifically accumulates in the acidic compartments and lysosomes, was significantly increased in XTC-1 in comparison to TPC-1 cells. Our hypothesis is therefore that an autophagic process might be constitutively activated in the oxyphilic cell line, perhaps as a result of the mitochondrial energetic impairment. To investigate this specific issue, we will determine the effect of specific inhibitors of autophagy, such as 3-methyladenine (10mM) and wortmannin (100nM) on both cell viability and ATP levels ofC6-ceramide-treated XTC-1 cells. Furthermore, the incorporation of the fluorescent dye monodansylcadaverine, a specific marker for autophagic vacuoles (7), will be utilized to quantify the changes occurring in these organelles during incubation with the ceramide analogue. These preliminary results suggest that XTC-1 cells incubated with C6-ceramide might represent a useful model with which to investigate the autophagic process.

1.Tallini G. (1998) Virchows Arch. 433:5-12.
2.Maximo V. et al. (2000) Virchows Arch. 437:107-115.
3.Maximo V. et al. (2002) Am J Pathol. 160:1857-1865.
4.Rogounovitch T. et al. (2004) Endocr J. 51: 265-277.
5.Savagner F. et al. (2001) Thyroid 11: 327-333.
6.Savagner F. et al. (2001) J Clin Endocrinol Metab. 86:4920-4925.
7.Munafò D.B. and Colombo M.I. (2001) J. Cell Sci. 114, 3619-3629

This work is supported by a grant from AIRC.

Regulation of ceramide metabolism in glioma cells: evidence on the role of phosphatidylinositol- 4-kinases.

T.Colleoni, L. Brioschi, P. Giussani, V. Anelli, R. Bassi, L. Riboni, G. Tettamanti and P.Viani
Dept. Medical Chemistry, Biochemistry and Biotechnology, Faculty of Medicine, University of Milan, Italy

A great number of evidence supports the role of ceramide (Cer), a key intermediate of sphingolipid metabolism as signalling molecule in mechanisms governing growth, differentiation and death in many cell types including glial cells. Different studies demonstrate that in glial cells Cer exerts antiproliferative and proapoptotic effects, and strongly support that Cer-signaling is altered in glial tumors. The control of Cer levels is a very complex process which involves both specific enzymes localized in different subcellular compartments and the regulation of Cer intracellular movements. Recent evidence suggests that the biological effects exerted by Cer depends on the variation of Cer levels in specific subcellular compartments, therefore correlated to the regulation of its intracellular traffic. Notwithstanding the mechanisms involved in Cer transport play a crucial role in Cer metabolism and signalling, the information on this process is very limited. Recently, a protein (CERT) that acts as ceramide carrier protein between endoplasmic reticulum (ER) and Golgi apparatus and is involved in sphingomyelin (SM) biosynthesis has been identified. The amino-terminal region of CERT contains a pleckstrin homology (PH) domain with selectivity towards phosphatidylinositol-4-phosphate (PI4P), to allow its targeting to the Golgi apparatus. In addition, recent evidence demonstrates that the interaction ofoxysterol-binding protein (OSBP) with ER and Golgi apparatus is regulated by its PH domain and is involved in the vesicle -mediated Cer transport from ER to the the Golgi for SM biosynthesis. This suggests the possibility that phosphoinositides, in particular PI4P a lipid known to be enriched in Golgi membranes, contributes to the regulation of ceramide metabolism and signalling. A number of different isoforms ofPI4-kinase (PI4-K) have been characterized. The isoenzymes differ from subcellular distribution and sensivity to inhibitors. Most of the isoenzymes are inhibited by phenylarsine oxide (PAO), whereas only the class IIIPI4-Ks, mainly associated to Golgi membranes, are sensitive to wortmannin (WT).

In the present study we investigate the effect of PI4-Ks on ceramide metabolism and traffic in C6 glioma cells. Treatment of C6 glioma cells with both PAO and WT results in a dose-dependent decrease of phosphatidylinositol phosphorylation. In pulse/chase experiments with 3H-sphingosine both inhibitors promote a dose-dependent increase of 3H-Cer with a concomitant decrease of 3H-SM and in a lesser extent of 3H-glucosylceramide. Metabolic experiments performed with N-hexanoyl-[3H]sphingosine and 3H-SM allow to exclude an effect of PI4-K inhibitors and/or polyphosphoinositides directly on SM-synthase, SMase orglucosylceramide-synthase activity. Moreover, PI4-K inhibitors strongly affect the intracellular distribution ofBODIPY-Cer. In fact in cells treated with WT and PAO the accumulation of the fluorescence in the perinuclear region representative of the Golgi apparatus was strongly reduced. Staining with NBD Cer a specific marker of the Golgi demonstrate the integrity of Golgi apparatus in WT and PAO treated cells.Taken together these results strongly suggest that the inhibition of a PI4-K localized in the Golgi apparatus impairs the biosynthesis of sphingomyelin and in a lesser extent of glucosylceramide, possibly reducing the dockng sites, namely PI4P, for ceramide targeting to the Golgi apparatus. This could represent a further element in the cross-talkbetween phosphoinositides and sphingolipid signaling involved in the control of glial cell fate.

Role of lipid molecules in tumor cell biology: old facts and new perspectives.

Ruggieri S.
Department of Experimental Pathology and Oncology, University of Florence, ruggieri@unifi.it, tel. 055 4282323, fax 055 4282333.

Studies of tumor lipids have developed in step with those regarding the role of lipid molecules in cell biology. The parallel development of these studies has represented an interesting example of a fruitful interaction in the investigation of cell physiology and tumor biology. In the sixties, the explosive interest in cholesterol regulation in the body in order to understand the pathogenesis of atherosclerosis led to the discovery that hepatomas lack the feedback inhibition of cholesterol synthesis. Much attention has been devoted to phospholipids in tumor cells, due to the recognition that phospholipids are essential membrane components involved in enzymatic activities, receptorial properties, cellular permeability — all of which are implicated in tumor cell biology. Structural analyses of phospholipids in different types of tumors revealed a profound alteration in their diacyl choline-glycerophospholipid molecular species. These analyses also showed an accumulation of ether-linked lipids in tumor cells, particularly evident in those with a high metastatic potential. This finding stimulated a great deal of research on the biosynthesis, distribution, and biological functions of ether-linked lipids.
Moreover, awareness that glycolipids and gangliosides are involved in cellular recognition, particularly in the altered social behavior of tumor cells, has opened the way to understanding the complexity of the family of glycolipid molecules, for which Hakomori’s great contribution should be aknowledged. Unlike phospholipids, a specific glycolipid pattern was not found in different tumors examined. In fact, the glycolipid biosynthesis exhibits a lineage-dependent specificity which leads to peculiarities in the glycolipid patterns in tumor cells of different origin. For instance, a reduction of the more complex gangliosides in virally-transformed cell lines has been reported by different laboratories, while an accumulation of fucose-containing glycolipids has been noticed in epithelial tumors of a different origin. Moreover, in a fibrosarcoma line, the metastatic properties appeared to be correlated with the content and cell surface expression of Gb3ose, a glycolipid characteristic of this line.
The tremendous expansion of research on inflammatory lipid mediators (platelet-activating factor, prostaglandins, thromboxanes, leukotrienes, hydroxyeicosatetraenoic, lipoxins) showed the importance of these mediators in several homeostatic reactions, including some which are implicated in various aspects of tumor progression, such as inflammatory processes, immunological responses and activation of platelets and endothelial cells. Several laboratories documented the involvement of arachidonic acid metabolites in growth rate and metastatic diffusion of tumor cells. Concentratingon the role played by platelet-activating factor (PAF) in certain steps of metastatic diffusion, we observed that: a) PAF is synthesized by different transformed cell lines under basal conditions and after stimulation with cytokines; b) PAF influences the interaction of melanoma and colon carcinoma cells with adhesive proteins of endothelial cells; and c) PAF promotes mobility of tumor cells by influencing the cytoskeleton.
The long-lasting consensus on the pro-tumoral activity of high fat diets focused attention on the different families (n-6, n-3) of polyunsaturated fatty acids (PUFA) as possible modulators of tumor progression. Indeed, contrasting effects on carcinogenesis and metastatic diffusion were observed in animals fed diets enriched with different families of PUFA : stimulatory with PUFA n-6, inhibitory with PUFA n-3.
A further insight on the role played by lipid molecules on carcinogenesis and tumor progression will be offered by the incoming investigation on the influence of lipid molecules on gene expression. This consideration is sustained by the recent report that the antitumoral effects of certain PUFA are mediated by peroxisome proliferator-activated receptors, a ligand-activated nuclear receptor superfamily which plays a regulatory role in several biological responses.

Biophysical properties of short- and long-chain ceramides.

Goñi, FM, Contreras, FX, Montes, LR, Sot, J and Alonso, A.
Unidad de Biofísica (CSIC-UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.

In the past decade the long-neglected ceramides (N-acylsphingosines) have become one of the most attractive lipid molecules in molecular cell biology, because of their involvement in essential structures (stratum corneum) and processes (cell signalling). Most natural ceramides have a long (16-24 C atoms) N-acyl chain, but short N-acyl chain ceramides (2-6 C atoms also exist in nature, apart from being extensively used in experimentation, because they can be easily dispersed in water. Long-chain ceramides are among the most hydrophobic molecules in nature, they are totally insoluble in water and they hardly mix with phospholipids in membranes, giving rise to ceramide-enriched domains. In situ enzymic generation, or external addition, of ceramides in membranes has at least three important effects: (i) lipid monolayer tendency to adopt a negative curvature, e.g. through a transition to an inverted hexagonal structure, is increased, (ii) bilayer permeability to aqueous solutes is notoriously enhanced, and (iii) transbilayer (flip-flop) lipid motion is promoted. Short-chain ceramides mix much better with phospholipids, promote a positive curvature in lipid monolayers, and their capacities to increase bilayer permeability or transbilayer motion are very low or inexistent.

Subcellular distribution of neutral ceramidase: localization to the outer mitochondrial membrane of rat liver.

*&°Porcelli AM, §Novgorodov SA, *Luberto C, &Rugolo M, §Obeid LM,and *Hannun YA.
*Department of Biochemistry and Molecular Biology and §Department of Medicine, MUSC, Charleston, SC (USA); &Dipartimento di Biologia E.S., Università di Bologna, Bologna, Italy.
°Via Irnerio 42, 40126, Bologna, Italy. Tel. 051/2091286; Fax 051/242576; Email: porcelli@alma.unibo.it

Ceramide, sphingosine and sphingosine-1P play important roles in the regulatory functions of cellular processes such as apoptosis, proliferation and differentiation. Ceramidases (CDases) are the enzymes that hydrolyze ceramide to free fatty acid and sphingosine, thus contributing to change the intracellular levels of these bioactive molecules. These enzymes have been classified in three groups: acid, neutral and alkaline. Although the acid and alkaline CDases appear to be clearly localized to the lysosomes and ER/Golgi, respectively (1-3), the intracellular localization of the neutral enzyme is not clear. In this regard we had noted from our own studies (4) and from published studies a significant variation in the subcellular localization of neutral CDase when overe-xpressed in various cell lines.
The aim of this work was to define the intracellular localization of endogenous CDase and toreconcile the results from tissue culture studies with subfractionation results. Rat liver subfractions were prepared using differential centrifugation and subsequent separation of pure mitochondria on Percoll gradient. CDase activity assay using D-e-C12-NBD-ceramide as substrate revealed that the activity was enriched in the pure mitochondrial fraction. Further, submitochondrial fractionation using digitonin showed that the enzyme resided preferentially in the outer mitochondrial membrane. Western blot analysis also supported these results and indicated that the molecular mass of neutral CDase in rat liver is around 85kDa. Unlike liver, most cell line are not very rich in mitochondria, and we hypothesized that whereas mitochondria may be enriched in the enzyme, over-expression may overwhelm the transfected cells. Therefore we investigatedthe subcellular localization of human neutral CDase in HEK 293 cells after transfectionwith GFP empty vector or vector in which GFP was at the C-terminus of human CDase. Subcellular fractionation of HEK cells showed that the specific activity of neutral ceramidase was highest in mitochondria. Nevertheless, mitochondria accounted for only a small fraction of the total activity. Confocal microscopy revealed that the over-expressed CDase exhibited a tubular pattern in the cytoplasm in close proximity to mitochondria and partially colocalized with an ER marker protein.
All together these results indicate that the endogenous neutral CDase is enriched in mitochondria from rat liver, and that the over-expression of the enzyme likely causes a mislocalization. This finding warrants caution in interpreting results from over-expression studies, in particular when applied to membrane-bound proteins.
References
Sugita M, Dulaney JT, Moser HW. Ceramidase deficiency in Farber’s disease. Science. 1972; 178:1100-1102.
Mao C, Xu R, Szulc ZM, Bielawska A, Galadari SH, Obeid LM. Cloning and characterization of a novel human alkaline ceramidase. J. Biol. Chem. 2001; 276:26577-26588.
Mao C, Xu R, Szulc ZM, Bielawska J, Becker KP, Bielawska A, Galadari SH, Hu W, Obeid LM. Cloning and characterization of a mouse endoplasmic reticulum alkaline ceramidase. J. Biol. Chem. 2003; 278:31184-31191.
El Bawab S, Roddy P, Quin T, Bielawska A, Lemasters JJ, Hannun YA. Molecular cloning and characterization of a human mitochondrial ceramidase. J. Biol. Chem. 2000; 275: 21508-21513.

Gangliosides are present in all subcellular fractions of rat liver.

¹Popa I, ²Ardail D, ¹Portoukalian J
¹Department of Dermatology, Edouard Herriot Hospital, 69437 Lyon Cx 03, France, ²Laboratory of Radiobiology, Lyon-Sud Medical School (Popa I, phone +33-4-72110607; fax +33-4-72110290; e-mail : popa@lyon.inserm.fr)

The site of biosynthesis of gangliosides has long been thought to be exclusively the Golgi apparatus and their localization to be mostly at the outer surface of plasma membranes. However, recent evidence suggest that the mitochondria-associated membrane (MAM) can also synthesize gangliosides. Moreover, the presence of gangliosides has been documented in mitochondria and nuclei. In order to gain insight on the presence of gangliosides in subcellular fractions, we have carried out analyses of the gangliosides isolated from highly purified subcellular fractionsof rat liver. The major gangliosides species of the total rat liver were GD1a>GM1>GM3>GD1b>GT1b>GD3>GM2. Gangliosides were identified by co-migration with standards and by immunostaining on thin-layer plates with specific antibodies. The subcellular fractions purified from rat liver were the following : whole mitochondria, mitochondrial outer and inner membranes, mitochondria-associated membranes (MAM), microsomes, Golgi apparatus, nuclei and plasma membranes. All subcellular fractions analyzed were found to contain gangliosides. The ganglioside contents and the quantitative patterns were different in each organite, but no qualitative differences could be observed. Our results show that gangliosides are present in all cellular membranes, but the respective patterns do not reflect the global ganglioside composition of a tissue.

Sphingomyelin metabolism changes after serum deprivation in neuronal cell nuclei.

Albi E¹, Cataldi S ¹, Bartoccini E¹, Mazzoni F ², Voccoli V³, Viola Magni M¹,Lazzarini R¹, and Garcia-GilM².
Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100, Perugia;2) Dpt. of Physiology and Biochemistry, University of Pisa, 3)Institute ofNeuroscience, CNR, Pisa.

We have previously shown that in murine embryonic hippocampal HN9 cells, serum deprivation induces variations in the nuclear enzyme activities involved in the sphingomyelin (SM) metabolism. Nuclear sphingomyelinase (SMase) increases biphasically, with an earlier peak one hour after serum deprivation, and a secondraise starting at 15 hrs. In contrast, nuclear sphingomyelin-synthase (SM-synthase) decreases after serum deprivation with a minimun after 1 hr. No variations of SM metabolism were observed in the homogenate. The aim of this work is to clarify if the enzymes present in the nuclei differ from those present in the homogenate on the basis of the physical chemical parameters of the SMase and the SM-synthase in homogenate and nuclei isolated from HN9 cells and so to ascertain the specificity of the above modificationsandevaluatethe SM and ceramide content changes in relation to the serum deprivation-induced apoptosis. 3H-PC and 14C-SM were used as precursors to evaluate the SM-synthase and SMase activityrespectively. The neutral SMase and SM-synthase have a pH optimum 7.2 in homogenate and 7.6 in nuclei, whereas the acid SMase, measured in the homogenate, has a pH optimum 5.00.Their activity is linear during the first 60 min and in the range from 50 to 200 mg protein. The enzyme activities obey a regular Michaelis-Menten kinetics in both preparations. The Km values of the neutral SMase are 1.48 x 10-3M for the homogenate and 1.26 x 10-4 M for nuclei whereas that of acid SMase is 4.55 x 10-5 M. The Vmax were 344, 113 and 183 pmol/mg protein/min respectively. The Km values of SM-synthase are 4.6 x 10-3M for homogenate and 1.59 x 10-4 for nuclei. The Vmax are 140 and 109 pmol/mg protein/min respectively. The physical chemical parameters of the nuclear enzymes in HN9 cells are similar to those reported previously for hepatocyte nuclei. The lower Kms of the nuclear SMase and SM-synthase indicate a higher affinity for their substrate compared to those of the homogenate. Our results suggest that the enzymes present in the homogenate and nuclei are different. The modifications of nuclear levels of SM and ceramide during the first hours of serum deprivation were studied by labeling the cells with 3H palmitic acid and measuring 3H-SM and 3H-ceramide in the purified nuclei. We have found that aftertwo hours of serum deprivation, the nuclear 3H-SM decreases while 3H-ceramide increases significantly, in agreement with the increase of SMase activity and inhibition of SM-synthase activity inhibited as previously reported.

Development of a high throughput screening assay for ceramidases and application to the screening of combinatorial libraries of sphingolipid analogs.

Bedia C, Triola G, Badalassi F, Grijalvo S, Casas J, Delgado A, Llebaria A and Fabriàs G.
Research Unit on BioActive Molecules (RUBAM), Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18. 08034-Barcelona. Spain. – FAX: 34-93-2045904 Phone: 34-93-4006115

The first step in the catabolic route of ceramide is its amide hydrolysis by ceramidases. Several types of ceramidases have been reported that differ in their optimum pH, intracellular location and substrate specificity. Mutations in the acidic form gives rise to Farber disease (1), a rare lysosomal storage disorder for which there is no current therapy. A promising novel strategy that has afforded promising results in Gaucher and Fabry diseases is the active site directed chaperone approach. This strategy relies on the use of competitive enzyme inhibitors at sub-inhibitory concentrations to aid the correct folding of the mutated enzyme, thus favouring its transport to the lysosome and increasing its residual activity. Although several inhibitors of ceramidases have been reported (2-4), potent inhibitors of the acidic enzyme are not available. The only acid ceramidase inhibitor so far described is N-oleoylethanolamine, and its potency is rather low (4). In contrast, potent and selective inhibitors of mechanistically similar hydrolases are known (5). In this context, we have recently undertaken a project aimed at the synthesis of combinatorial libraries of sphingolipid analogs with a general structure combining the diverse structural motifs present in reported inhibitors of ceramidases and mechanistically similar amide hydrolases. To identify and select competitive inhibitors within the combinatorial lipid libraries, the development of a high throughput screening (HTS) procedure was necessary.
In this work we describe the development of a HTS procedure to determine ceramidase activity and its application to the screening of combinatorial libraries of lipid analogs. As detailed in the figure, the method is based on the Reymond’s concept (6), which consists on the use of a fluorogenic substrate, which, upon amide hydrolysis, affords an aminoalcohol. Its oxidation with NaIO4 gives rise to an aldehyde, which is broken down to fluorescent coumarine in BSA at pH = 8.0.
Missing figure

The fluorescent substrate can be easily prepared as outlined in the scheme.

As depicted in the figure, experiments with the neutral ceramidase have shown that compound CHA is a substrate of this enzyme with a Km of 120 µM and a Vmax = 212 nmole/min. Using this fluorimetric assay, combinatorial libraries of sphingolipid analogs can be screened and ceramidase inhibitors can be identified

References
1. He X, Okino N, Dhami R, Dagan A, Gatt S, Schulze H, Sandhoff K, Schuchman EH.J. Biol. Chem. 2003;278:32978-32986.
2. Bielawska A, Greenberg MS, Perry D, Jayadev S, Shayman JA, McKay C, Hannun YA. J. Biol. Chem. 1996;271:12646-12654.
3. Selzner M, Bielawska A, Morse MA, Rudiger H. A, Sindram D, Hannun YA, Clavien PA Cancer Res. 2001;61:1233-1240.\
5. Boger DL, Sato H, Lerner AE, Hedrick MP, Fecik RA, Miyauchi H, Wilkie GD, Austin BJ, Patricelli MP, Cravatt BF. Proc. Natl. Acad. Sci. USA. 2000;97:5044-5049.
6. Wahler D, Badalassi F, Crotti P, Reymond, JL. Chemistry–A European Journal. 2002;8:3211-3228.

Double strand RNA and sphingomyelin.

Rossi G., Viola Magni MP, Albi E.
Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100 Perugia, Italy. e-mail: ispatgen@unipg.itealbi@unipg.it

A complex formed by 97% protein, 1.7% RNA, 0.4% DNA and 0.18% phospholipids (PLs), constituted by sphingomyelin (SM) and phosphatidylcholine (PC), was isolated from hepatocyte nuclei (1). The RNA appeared to be double-strand since was resistant to RNase treatment and became sensitive to enzymatic hydrolysis when submitted to thermal denaturation. The digestion of SM with sphingomyelinase (SMase) trasformed Rnase-undigested RNA in RNase-sensitive RNA, thus suggesting that the SM may form a bridge between two RNA strands (1). The EM analysis, using double labeling with anti-RNA polymerase II antibody and N-SMase-gold complex showed that SM colocalizes with RNA-polymerase II in the transcription sites (unpublished data). The aim of this work was to highlight the relationship between SM and RNA transcription in the intranuclear complex. Hepatectomised animals were killed 24 hrs after operation, 100mCi 3H-uridine was injected 1h prior to killing. The 3H-uridine incorporation value was 1893 cpm/mg RNA in the homogenate and increased by 2.9 and 3.7 times in the nuclei and in the intranuclear complex respectively. The data suggest that the RNA present in the complex is neosynthesised. To investigate if the RNA was synthesised directly in the intranuclear complex, the possible presence of a transcription factor STAT3 was detected by immunoblotting with specific antibodies. The results showed that the STAT3 is present in the intranuclear complex, its molecular weight is 180 KDa which is a dimeric active structure formed in the cytoplasm and transferred in the nucleus. After SMase treatment the dimeric structure decreased strongly and a spot corresponding to 80 KDa, a monomeric form, appeared. It is possible that SM and STAT3 interact in the transcription process and facilitate the formation of the RNA loops. The SMase degrades STAT3 and SM favouring the splicing process.
1) Micheli,M., Albi,E, Leray,C., and Viola Magni,M.P. “Nuclear sphingomyelin protects RNA from RNase action” FEBS Letters 431, 443-447, 1998.

Sphingosine kinase as a ‘sensor’ during chemotherapy-induced apoptosis in prostate cancer.

Cuvillier O¹, Pchejestki D¹,Bonhoure E¹, Levade T¹, Calvet C², Malavaud B^2  
1 Inserm U466 and ² Urology Department, Rangueil Hospital, 1 avenue Poulhes, 31059 Toulouse Cedex 9, France
Tel : (+33) 561.32.20.60 ; Fax : (+33) 561.32.20.84

Prostate cancer is the most common malignancy and the second leading cause of cancer death in men, killing more than 200,000 men annually worldwide. Many forms of prostate cancer initially are androgen dependent, but the response to androgen-ablation therapy is transient, and after a few years, the majority of prostate cancers relapse to the status of androgen independence resulting in death. Despite the availability of various therapeutic approaches (ionizing radiation, chemotherapy) that kill tumor cells by apoptosis, many cancer cells develop resistance to apoptosis.
Sphingolipid metabolites – including ceramide, sphingosine and sphingosine 1-phosphate (S1P)- are active mediators that play essential roles in cell growth, survival and death. Abundant evidence suggests that ceramide is a critical messenger of ionizing radiation- and chemotherapeutic drug-induced apoptosis. In contrast to the growth-inhibitory and pro-apoptotic effects of ceramide, S1P, which is produced by phosphorylation of sphingosine by the oncogenic sphingosine kinase, has been implicated in cell growth and inhibition of ceramide-mediated apoptosis. It has been hence suggested that the dynamic balance between levels of ceramide versus S1P, and consequent regulation of opposing signaling pathways, is an important factor that could determine whether a cell survives or dies.
Herein, we report that sphingosine kinase could act as a ‘sensor’ during chemotherapy-induced apoptosis in prostate cancer. In other words, we have found that the chemosensitivity/chemoresistance status is well correlated with sphingosine kinase activity. In prostate cancer cells in which chemotherapeutics induced a strong apoptosis, sphingosine kinase activity was rapidly down regulated. Accordingly, levels of endogenous ceramide and S1P were respectively increased and diminished. Conversely, there were no changes in sphingosine kinase activity (nor in ceramide and S1P levels) in tumor cells in which chemotherapeutics have no or little effect on cell viability. To demonstrate that sphingosine kinase inhibition was instrumental for chemotherapeutic-induced apoptosis, we over expressed its gene in two prostate cancer cell lines.As anticipated, sphingosine kinase overexpression markedly protected cells from chemotherapeutic-induced apoptosis.
Ability of sphingosine kinase to determine the resistance of cancer cells to chemotherapy might propose inhibition of this enzyme for potential application in cancer treatment. Strategies that elevate cellular ceramide and diminish S1P by switching off sphingosine kinase could be used for therapies aimed to arrest growth or promote apoptosis. To this end, we have utilized newly developed sphingosine kinase inhibitors to alter this signaling pathway. These inhibitors could successfully overcome chemoresistance by triggering apoptosis in the prostate cancer cell lines used in this study.
By its very nature, the sphingolipid biostat is poised for pharmacologic manipulation, and we can anticipate therapeutic success from the thoughtful use of molecules that can interfere with it by inhibiting the key enzyme, sphingosine kinase.

Sphingosine 1 Phosphate (S1P) protects mice from the radiation–induced GI syndrome and LPS-induced death.

¹Bonnaud S, ¹Niaudet C, ²Fuks Z, ²Kolesnick R, ¹Paris F.
¹INSERM UMR601, Nantes, France and ²Memorial Sloan-Kettering Cancer Center, New York, NY. Paris F. INSERM UMR601, IFR26, 9 quai Moncousu, 44000 Nantes, France. Tel : +33.(0)2.40.08.47.47 Fax : +33.(0)2.40.35.66.97

While ceramide is considered a pro-apoptotic factor, its metabolite, S1P, acts as a proliferative, anti-apoptotic factor. Recent results showed that acid sphingomyelinase gene disrupted (asmase-/-) oocytes and S1P treated oocytes resist radiation-induced apoptosis ex vivo (1). Moreover, in adult mice, oocyte loss, ovarian aplasia and sterility, observed in 0.1 Gy irradiated wild-type mice, were prevented by injection of S1P into the ovary prior to radiation (2). This study showed that S1P might be a promising agent for the protection of critical tissues against toxicity of anticancer drugs. The gastrointestinal (GI) syndrome is a common side effect of anticancer treatments and limits their efficacy. Our previous data showed that microvascular endothelial apoptosis after whole body irradiation at 15 Gy leads to stem cell dysfunction, crypt damage, organ failure, and death from the GI syndrome (3). These events were prevented when endothelial apoptosis was inhibited pharmacologically, by i.v. basic fibroblast growth factor (bFGF) injection, or genetically, by deletion of asmase. bFGF-treated mice or asmase-/- mice died later from bone marrow aplasia. We now report that retro-orbital injection of S1P in wild-type C57Bl/6 mice, prior to irradiation, mimics the effect of asmase disruption. S1P protected mice from death caused by the GI syndrome. Survival of 15 Gy-irradiated mice after S1P treatment was increased (median survival 9 days vs. 6 days for 15 Gy control mice; p<0.05). Autopsies showed that 80% of 15 Gy irradiated animals treated with S1P died from marrow failure rather than the GI syndrome. Furthermore, published data already showed that LPS-induced mouse death by a disseminated form of endothelial apoptosis was mediated through ceramide generation, and inhibitable by bFGF injection or asmase deletion (4). Similarly, S1P-pretreated mice were protected from lethal LPS injection (76% of mice survive 175 mg LPS/25g body weight after S1P pretreatment vs. 19% of control mice; p<0.05). In vivo investigations showed that S1P radioprotection against tissue damage and death in these models is due to direct inhibition of endothelial cell apoptosis.
In order to better describe the molecular pathway used by S1P to inhibit endothelial cell apoptosis, we decided to study the effect of S1P in irradiated microvascular endothelial cells (HMEC-1). We first showed that a 15 Gy irradiation kills 40% of cells by apoptosis after 24 hours. Pretreatment with 1mM S1P 2 hours before irradiation was able to inhibit apoptosis by 50% in HMEC-1 cells. In order to know whether the S1P was acting directly against ceramide generation or through S1P receptors and G protein activation, we pretreated endothelial cells with an inhibitor of Gi/G0 proteins activation, the Pertussis Toxin (PTX), before S1P treatment and irradiation. This PTX treatment reverted radioprotection of S1P in HMEC-1 cells. In vivo investigation are underway to determine whether PTX pretreament can block the radioprotection of the microvascular endothelium, tissue damage and death caused by S1P.
References
1. Morita Y, Perez GI, Paris F, Miranda SR, Ehleiter D, Haimovitz-Friedman A, Fuks Z, Xie Z, Reed JC, Schuchman EH, Kolesnick RN, Tilly JL. Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy. Nature Medicine. 2000;6(10):1109-14.
2. Paris F, Perez GI, Fuks Z, Haimovitz-Friedman A, Nguyen H, Bose M, Ilagan A, Hunt PA, Morgan WF, Tilly JL, Kolesnick R. Sphingosine 1-phosphate preserves fertility in irradiated female mice without propagating genomic damage in offspring. Nat Med. 2002 Sep;8(9):901-2. Nature Medicine. 2002;8(11):1329
3. Paris F, Fuks Z, Kang A, Capodieci P, Juan G, Ehleiter D, Haimovitz-Friedman A, Cordon-Cardo C, Kolesnick R. Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science. 2001;293(5528):293-7.
4. Haimovitz-Friedman A, Cordon-Cardo C, Bayoumy S, Garzotto M, McLoughlin M, Gallily R, Edwards CK 3rd, Schuchman EH, Fuks Z, Kolesnick R. Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation.J Exp Med. 1997;186(11):1831-41.

Sphingosine-1-phosphate signalling in neuronal cells – Studies with a synthetic analogue.

van Echten-Deckert G and Nätzker S
Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
phone 0049-228-732703; fax 0049-228-737778; e-mail: g.echten.deckert@uni-bonn.de

cis-4-Methylsphingosine is a synthetic cell permeable pro-drug, that is taken up by cells and phosphorylated to a metabolically stable cis-4-methylsphingosine-1-phosphate (1). This compound was shown to mimic the mitogenic effect of its short living physiological counterpart sphingosine-1-phosphate (S1P) in Swiss 3T3 fibroblasts (2). In neuroblastoma cells, however, cis-4-methylsphingosine-phosphate was found to induce apoptosis and this effect was mediated by an opposite regulation of p38 and of ERK MAPKs (3).
We have now investigated the effect of both, S1P and its synthetic analogue in highly differentiated primary cultured neurons. In contrast to S1P, which had no effect on growth of these post-mitotic cells, cis-4-methylsphingosine-1-phosphate induced apoptosis. Like in neuroblastoma cells MAPK pathways were involved in this process. In addition, however, an aberrant activation of the cell cycle machinery was found to be required.
Interestingly, our results document that the different physiological effects, apoptosis in case of the accumulating metabolically stable synthetic compound versus no apoptosis in case of the short-living S1P, rely only on nuances of impact. In other words – similar pathways are affected by both compounds albeit in a sustained and more pronounced manner in case of the metabolically stable synthetic compound.
References
1 – van Echten-Deckert G, Zschoche A, Bär T, Schmidt RR, Raths A, Heinemann T, Sandhoff K. cis-4-Methylsphingosine decreases sphingolipid biosynthesis by specifically interfering with serine palmitoyltransferase activity in primary cultured neurons. J. Biol. Chem. 1997;272:15825-33.
2 – van Echten-Deckert G, Schick A, Heinemann T, Schnieders B. Phosphorylated cis-4-methylsphingosine mimics the mitogenic effect of sphingosine-1-phosphate in Swiss 3T3 fibroblasts. J. Biol. Chem. 1998;273:23585-89.
3 – Nätzker S, Heinemann T, Figueroa-Perez S, Schnieders B, Schmidt RR, Sandhoff K, van Echten-Deckert G. cis-4-Methylsphingosine-phosphate induces apoptosis in neuroblastoma cells by opposite effects on p38 and ERK MAPKs. Biol. Chem. 2002;383:1885-94.

Sphingosine kinase activity is required for sphingosine-mediated phospholipase d activationin C2C12 myoblasts.

Meacci E, Donati C, Cencetti F, Nuti F, Becciolini L and BruniP
Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze,Viale G.B. Morgagni 50, 50134 Firenze; Istituto Interuniversitario di Miologia (IIM); tel ++39055413765; fax ++390554222725

Sphingosine (Sph) has been implicated as modulator of membrane signal transduction systems and as regulatory element of cardiac and skeletal muscle physiology but scarce information is presently available on its precise mechanism of action. Recent studies have shown that sphingosine 1-phosphate (S1P), generated by sphingosine kinase (SphK) action on Sph, possesses also biological activity, acting as intracellular messenger as well as extracellular ligand for specific membrane receptors. At present however, it is not clear whether biological effects elicited by Sph are attributable to its conversion into S1P. In the present study this issue has been investigated.
C2C12 cells were routinely grown in DMEM supplemented with 10% fetal calf serum in an atmosphere of 95% air 5% CO2. For their differentiation into myotubes confluent cells were shifted to low serum medium (DMEM added with 2% horse serum). Mature phenotype was obtained after 5-6 days. Initially, sphingosine (Sph) was examined for its ability to activate phospholipase D (PLD), as observed in other cell types (1). Indeed, 1mM Sph appreciably stimulated PLD activity. Intriguingly, stimulation of PLD by Sph was found to be inhibited by pertussis toxin (PTx), as also observed for S1P-induced PLD activation (2) and could not be detected in differentiated C2C12 cells, in which S1P is uncoupled from PLD stimulation (3), supporting the hypothesis that a common pathway accounts for the action of both sphingoid bases. Furthermore, experiments performed in C2C12 myoblasts overexpressing S1P2 or in which endogenous receptor was down-regulated by specific antisense oligonucleotides, were in favour of a major role of S1P2 in the activation of PLD by Sph. Moreover, Sph-induced activation of PLD was inhibited by N,N-dimethyl-D-erythro-sphingosine (DMS), at concentrations capable to block specifically SphK. The crucial role of SphK-derived S1P in the activation of PLD by Sph was confirmed by the observed potentiated effect of Sph in myoblasts where SphK1 was overexpressed and the attenuated response in cells transfected with the dominant negative form of SphK1. Notably, the in vivo measurement of S1P formation by employing labelled ATP revealed that cell-associated SphK active in the extracellular compartment largely contributed to the transformation of Sph into S1P, being negligible the amount of SphK released into the medium. It will be important to establish whether the here identified mechanism of action is implicated in the multiple biological effects elicited by Sph in muscle cells.
Spiegel S and Milstien S. Sphingoid bases and phospholipase D activation. Chem. Phys. Lipids 1996;80: 27-36.
Meacci E, Vasta V, Donati C, Farnararo M, Bruni P Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C. FEBS Lett. 1999; 457: 184-188.
Meacci E, Cencetti F, Donati C, Nuti F, Farnararo M, Kohno T, Igarashi Y, Bruni P. Down-regulation of EDG5/S1P2 during myogenic differentiation results in the specific uncoupling of sphingosine 1-phosphate signalling to phospholipase D. Biochim Biophys Acta 2003; 1633:133-142.

Involvement of sphingosine in glucocorticoid-induced apoptosis of thymocytes.

Lépine S,* Lakatos B,*† Courageot M-P,* Le Stunff H,§ Sulpice J-C,* Giraud* F.
*Biomembranes et Messagers Cellulaires and §Activation Cellulaire et Transduction des Signaux,CNRS UMR8619,Université Paris XI-Orsay, France; †present address: Department of Biochemistry and Microbiology, Slovak University of Technology, Bratislava, Slovak Republic.

Corresponding author: Dr. Françoise Giraud, Biomembranes et Messagers Cellulaires, Bat 440, UniversitéParis XI-Orsay, 91405 Orsay Cedex, France. Phone: 33-1-69 15 76 44, Fax: 33-1 69 15 49 61, E-mail: francoise.giraud@ibaic.u-psud.fr

During the selection process in the thymus, most thymocytes are eliminated by apoptosis through signaling via TCR or glucocorticoids. The involvement of ceramide (Cer) and sphingosine (SP), important apoptotic mediators, remains poorly defined in glucocorticoid-induced apoptosis. We report that, in mouse thymocytes, apoptosis triggered by 10-6 M dexamethasone was preceded by a caspase-dependent Cer and SP generation, together with activation of acidic and neutral ceramidases. Apoptosis was drastically reduced by blocking either sphingolipid production (by acid sphingomyelinase inhibitor) or SP production (by ceramidase inhibitors), but not by inhibition of de novo Cer synthesis. Thus, SP generated through acid sphingomyelinase and ceramidase activity would contribute to the apoptotic effect of dexamethasone. Consistent with this hypothesis, SP addition or inhibition of SP kinase induced thymocyte apoptosis. Dexamethasone induced a proteasome-dependent loss of mitochondrial membrane potential DYm and caspase-8, -3 and -9 processing. Apoptosis was abolished by inhibition of DYm loss or caspases-8 or -3, but not caspase-9. DYm loss was independent of SP production and caspase-8, -3 and -9 activation. However, inhibition of SP production reduced caspase-8 and -3, but not caspase-9 processing. Proteasome inhibition impaired activation of the three caspases, whereas inhibition of DYm loss solely blocked caspase-9 activation. These data indicate that dexamethasone-induced apoptosis is mediated in part by SP which contributes, together with proteasome activity, to caspase-8/-3 processing independently of mitochondria, and in part by the proteasome/mitochondria pathway, although independently of caspase-9 activation.

Acid ceramidase controls daunorubicin resistance of human hepatoma cells via the ceramide/sphingosine-1-phosphate switch.

Morales A¹, París R¹, Marí M¹, Villanueva A², García-Ruiz C¹, 3 and Fernández-Checa JC 1, 3
¹ Liver Unit, Institut de Malalties Digestives, Hospital Clínic i Provincial, Institut d’Investigacions Biomèdiques August Pi i Sunyer, ² Institut Català d’Oncologia, Hospital Duran i Reynals, Granvia Km 2.7, 08907 Hospitalet, and ³Departmento de Patología Experimental, Instituto Investigaciones Biomédicas Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, 08036, Spain. Correspondence JC F-Ch (Liver Unit, Hospital Clinic, C/Villarroel, 170, 08036-Barcelona, checa229@yahoo.com).

Sphingolipids comprise a family of bioactive lipids that have been described to regulate and mediate diverse cellular functions with opposing outcomes such as in cell proliferation and growth arrest (1-3). In this regard, ceramide and sphingosine-1-phosphate (S1P), play an antagonizing role in cell death. Since ceramidases contribute to the conversion of ceramide to S1P, we analyzed the role of acid ceramidase in the response of tumor cells to chemotherapy. We report that daunorubicin (DNR) selectively stimulated acid ceramidase, decreasing the cellular ceramide to S1P balance. Acid ceramidase silencing by small interfering RNA or inhibition by N-oleoylethanolamine (NOE) enhanced the ceramide/S1P ratio, sensitizing human hepatoblastoma cell lines HepG2, SK-Hep, Hep-3B and human colon HT-29 cells to DNR-induced apoptosis. The sensitisation to DNR by NOE was reversed by extracellular S1P via a pertussis toxin-independent mechanism. The combination of DNR plus NOE resulted in ultrastructural changes in mitochondria, triggering reactive oxygen species generation, cytoplasmic release of Smac/DIABLO and cytochrome c, and caspase-3 activation. Importantly, the apoptotic potential of DNR plus NOE was selective for liver tumor cells, as primary hepatocytes were insensitive to DNR with or without NOE. Thus, acid ceramidase mediates the resistance of tumor cells to DNR through the sphingolipid ceramide/S1P rheostat and hence its antagonism may be of therapeutic value.
References
1. Obeid, LM, Linardic CM, Karolak LA, Hannun YA.Programmed cell death induced by ceramide. Science.1993. 1769-1771.
2. Hannun YA, and Obeid LM The Ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J. Biol. Chem. 2002. 277:25847-25850.
3. Spiegel S, andMilstein S. Sphingosine-1-phosphate: an enigmatic signaling lipid. Nat. Rev. Mol. Cell Biol. 2003. 4:397-407.

Biological activity of resveratrol via ceramide signaling.

P. Signorelli, R. Ghidoni
Laboratory of Biochemistry and Molecular Biology San Paolo Medical School, Milan, Italy – via A. di Rudinì, 8 – 20142 Milan – paola.signorelli@unimi.it

Resveratrol (3,4′,5-trans-trihydroxystilbene) is a polyphenol small molecule, naturally occurring in a variety of dietary vegetables (roots, peanuts, grape skin) and produced by plants in response to external stresses such as UV and pathogens attack. In human cells, resveratrol treatment affects a variety of cellular targets and the compound acts as anti-oxidant, radical scavenger, anti-inflammatory and antitumoral agent. Plants produce resveratrol to eliminate injured cells via an apoptotic mechanisms; similarly resveratrol induces apoptosis in human cancer cells, in a dose dependant fashion. We demonstrated that in human tumors of different origins (mammary adenocarcinoma, prostate carcinoma and melanoma) resveratrol treatment induces accumulation of endogenous ceramide. Ceramide formation is associated with an increased rate of sphingomyelin hydrolysis at plasmamembrane (nSMase) and of de novo synthesis of sphingolipids. Impairing ceramide accumulation, via metabolic inhibitors, rescued from resveratrol induced cell death. Thus ceramide is a necessary mediator of resveratrol-induced apoptotic mechanism. Since many chemotherapic agents that are effective in cell culture do not show equal efficacy in vivo, due to intrinsic resistance and reduced exposure to drugs of a solid tumoral mass, we tested resveratrol effect on a three dimensional culture of mammary adenocarcinoma cells. Resveratrol determined impairment of three-dimensional growth, disruption of a grown mass of tumoral cells and death of dispersed cells. Moreover the remaining aggregated cells were not able to proliferate if isolated and re-seeded (colony formation), showing a reduced metastasising activity. Even in three-dimensional culture resveratrol induced ceramide intracellular accumulation that is dose dependent and parallels the cytostatic and cytotoxic effect of the compound. In order to investigate on structure-function relation, resveratrol analogues were administered to prostate cancer cells. Whereas trans-stilbene (no hydroxylated) had no effect on cell proliferation, survival and induction of ceramide intracellular increase, the substitution of methoxyl groups in lieu of hydroxyl-groups resulted in an extremely toxic compound inducing cell death by a non apoptotic mechanism and unable to increase ceramide intracellular content. Piceatannol (3,4,3′,5′-trans-tetrahydroxystilbene), another analogue with an additional hydroxyl group, showed biological effects comparable to resveratrol. These results assess that resveratrol antitumoral activity is related to its phenolic moiety that targets, either directly or indirectly, ceramide metabolism.

De novo-synthesized ceramide is involved in cannabinoid-induced apoptosis of LeukAemia cell lines.

Herrera B, Carracedo A, Gómez del Pulgar T, Guzmán M and Velasco G
Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain
Fax: +34 913944672; Phone: +34 913944668; e-mail: blancahg20@hotmail.com

Cannabinoids, the active components of Cannabis sativa and their derivatives, exert a wide spectrum of effects both in the central nervous system and in peripheral locations due to their ability to bind to the specific CB1 and CB2 cannabinoid receptors.
During the last few years, research from several laboratories has ravelled the therapeutic potential of cannabinoids as antitumoral agents. However, the mechanisms involved in these antiproliferative actions have been only partially elucidated. Interestingly, cannabinoid-triggered ceramide accumulation has been identified as one of the mechanisms involved in the antitumoral actions of these compounds in gliomas.
Here, we employed the human leukaemic cell lines Jurkat and SupT1 -that had been previously shown to undergo apoptosis in response to cannabinoids- to gain insight into the intracellular pathways leading to cannabinoid-induced apoptosis of tumoral cells. For this purpose, cells were incubated with cannabinoids in the presence or absence of several inhibitors and antagonists, and cell viability, apoptosis, ceramide levels and serine palmitoyltransferase activity were determined. Western blot analyses were performed to measure expression, distribution and phosphorylation of several proteins.
Challenge of Jurkat and SupT1 cells to different cannabinoid receptor agonists and antagonists show that apoptosis occurs via CB2 receptor. Regarding intracellular signaling, data show that: (i) the pro-apoptotic effect of cannabinoids was prevented by pharmacological abrogation of the ceramide synthesis de novo pathway; (ii) cannabinoid treatment triggered a rapid accumulation of ceramide and a parallel stimulation of serine palmitoyltransferase activity; (iii) activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase was observed after cannabinoid challenge; (iv) cannabinoid treatment led to cytochrome c release from mitochondria and to activation of caspase 3.
Taken together, these data provide clues about the mechanism of cannabinoid pro-apoptotic action, and point to the involvement of de novo-synthesized ceramide in the process of CB2-induced apoptosis of human leukaemia cells.

Frataxin prevents ceramide-induced apoptosis.

Ventura N*, Condò I*, Malisan F and Testi R $
equally contributed to this work
Laboratory of Immunology and Signal Transduction, Department of Experimental Medicine,
University of Rome “Tor Vergata”. Via Montpellier, 1. 00133 ROMA.
Tel: 0672596502- Fax: 0672596505

Defective frataxin expression in humans causes Friedreich’s Ataxia (FA), an autosomal recessive neurodegenerative syndrome characterized by progressive ataxia, dysarthria, skeletal abnormalities, glucose intolerance and cardiomyopathy. Frataxin is a highly conserved mitochondrial protein involved in the biogenesis of iron/sulfur clusters (ISC) and iron homeostasis. Low frataxin levels affect mitochondrial energy metabolism as a consequence of altered biogenesys of ISC enzymes, which include several components of the mitochondrial electron transport chain.
FA cells are more sensitive to oxidative stress, yet the involvement of frataxin in cell survival is still obscure. Herein, we show that the transient overexpression of frataxin prevents ceramide-induced ROS accumulation and apoptosis in human tumor cells, with the same efficiency of the anti-apoptotic protein Bcl-2. GD3-mediated apoptosis, driven by GD3 synthase overexpression, is also inhibited by co-transfection of the cells with frataxin. Moreover, differently from Bcl-2, frataxin protects against anti-Fas induced apoptosis. Accordingly, frataxin-deficient FA cells are more sensitive to Fas, ceramide- and GD3-induced cell death. Taken together, these results suggest a protective role of frataxin in apoptosis.

Regulation of macroautophagy by ceramide in mammalian cells.

Scarlatti F.¹,2, Bauvy C.¹, Ghidoni R.² and Codogno P.^1
1INSERM U504, 16 avenue Paul Vaillant Couturier, 94807 Villejuif Cedex, France. Patrice Codogno Tel: 33145595042, Fax: 33146770233, e-mail: codogno@vjf.inserm.fr
²Laboratory of Biochemistry and Molecular Biology San Paolo Medical School, via Rudini 8, 20142 Milan Italy.

Macroautophagy is an evolutionary conserved lysosomal pathway involved in the turnover of long-lived proteins and organelles (1). Macroautophagy starts with the formation of a multilayer membrane bound autophagosome that sequesters fractions of the cytoplasm. In mammalian cells, most of autophagosomes receive inputs from endocytic compartments before fusing with lysosomes where the degradation of the sequestered material is completed. The discovery of a family of genes (ATG: AuTophaGy-related genes. Reviewed in Refs. 2,3) conserved from yeast to humans, and involved in the formation of autophagosomes has shed light on the importance of macroautophagy in physiology (adaptation to starvation, development and longevity) (4) and pathophysiology (cancer, myopathies, neurodegeneratives diseases) (5). The elucidation of the molecular control of autophagy will also lead to a better understanding of the role of macroautophagy during cell death (6). As a great number of extracellular stimuli (starvation, hormonal or therapeutic treatment) and intracellular stimuli (accumulation of misfolded proteins, invasion of microorganisms) is able to modulate the autophagic response, it is not surprising that several signaling pathways are involved in the control of macroautophagy. The class I PI3K/PKB/mammalian Target of Rapamycin (mTOR) signaling pathway plays a major role in transmitting autophagic stimuli because of its ability to sense nutrient, metabolic and hormonal signals (7).
Macroautophagy is under the control of different classes of phosphatidylinositol 3-kinases (PI3Ks). 3-methyladenine inhibits the formation of autophagosomes by interfering with the activity of the class III PI3K suggesting that the production of PtdIns3P is required during early steps of macroautophagy (8). By contrast the stimulation of the activity of class I PI3K inhibits the autophagic pathway. The production of PtdIns(3,4)P2 and PtdIns(3,4,5)P3recruits the serine/threonine kinase Akt/protein kinase B (PKB) and its activator the kinase PDK1. This signaling pathway which is involved in many cellular processes including proliferation and cell survival is negatively regulated by the tumor suppressor PTEN which dephosphorylates the D-3 position of PtdIns(3,4)P2 and PtdIns(3,4,5)P3. The overexpression of PTEN counteracts the down-regulation of macroautophagy by the stimulation of class I PI3K signaling pathway whereas the expression of a constitutively active form of PKB has an inhibitory effect on macroautophagy (9). More recently we have shown that the sphingolipid ceramide, which is involved many stress responses, upregulates macroautophagy (10). Exogenous C2-ceramide (C2-Cer) stimulates macroautophagy in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B1. Ceramide reverted the class I PI3K-dependent inhibition of macroautophagy, by interfering with the activation of PKB, and stimulated the expression of the autophagy gene beclin 1 (the ortholog of yeast ATG6) (11). Ceramide is also the mediator of the tamoxifen (TAM)-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells (12). Myriocin, an inhibitor of de novo ceramide synthesis abrogates the accumulation of autophagic vacuoles in TAM-treated MCF-7 cells. Interestingly, the glucosylceramide synthase inhibitor PDMP mimicked the TAM-dependent accumulation of vacuoles. PDMP, TAM and C2-Cer stimulated the expression of beclin 1 whereas myriocin antagonized the TAM-dependent upregulation of beclin 1 and the PKB inhibition by ceramide.
In conclusion, macroautophagy is controlled by the interplay between protein and lipid signaling. The control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.
References
1. Klionsky, D. J., and Emr, S. D. (2000) Science 290, 1717-1721.
2. Ohsumi, Y. (2001) Nat. Rev. Mol. Cell Biol. 2, 211-216.
3. Klionsky et al. (2003) Dev. Cell 5, 539-545.
4. Levine, B., and Klionsky, D. J. (2004) Dev. Cell 6, 463-477.
5. Cuervo, A. M. (2004) Trends Cell Biol. 14, 70-77.
6. Gozuacik, D., and Kimchi, A. (2004) Oncogene 23, 2891-2906.
7. Codogno, P., and Meijer, A. J. (2004) in Autophagy (Klionsky, D. J., ed), pp. 26-48., Landes Bioscience, Georgetown, TX.
8. Petiot et al.(2000) J. Biol. Chem. 275, 992-998.
9. Arico et al. (2001) J. Biol. Chem. 276, 35243-35246.
10. Scarlatti et al. (2004) J. Biol. Chem. 279, 18334-18391.
11. Liang et al. (1999) Nature 402, 672-676.
12 Bursch et al. (2000) J. Cell Sci. 113, 1189-1198.

Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa.

Tuson M, Marfany G, Gonzàlez-Duarte R.
Departament de Genètica. Facultat de Biologia. Universitat de Barcelona, Av. Diagonal 645, E-08028-Barcelona, Catalonia, Spain – Phone: +34934021500. Fax: +34934110969. E-mail: rgonzalez@ub.edu

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease characterized by the progressive attrition of mature photoreceptor cells through apoptosis, and which eventually results in complete blindness in early adulthood. At present, 38 genes and loci have been implicated in this monogenic, albeit complex, disorder, emphasizing the high genetic heterogeneity of RP. These include genes encoding proteins of the phototransduction cascade, proteins of the visual cycle, proteins responsible for the structure and polarity of the photoreceptors, and regulatory proteins, such as transcription and splicing factors (1). To date, none of the described RP genes have been directly involved in the apoptotic pathways that lead to photoreceptor loss. In the present study, we report a novel RP gene encoding a ceramide kinase enzyme (CERKL), which is responsible for the disease in a Spanish consanguineous family with autosomal recessive retinitis pigmentosa (arRP). Previous linkage analysis in this family allowed us to define a new arRP locus (RP26) within an 11-centiMorgan (cM) interval (17.4 Megabases, Mb) on 2q31.2-q32.3 (2). We further refined the RP26 locus down to 2.5 Mb by microsatellite and single-nucleotide polymorphism (SNP) homozygosity mapping. This 2.5-Mb candidate region harboured nine genes, which were analyzed for mutations, but none showed any pathogenic variant in the patients of this family. Subsequently, an exhaustive gene search based on expressed-sequence-tag (EST) data unveiled two partial sequences corresponding to a novel gene. This new gene spanned 13 exons and encoded a predicted protein of 532 amino acids that shared the highest similarity (29% identity; 50% similarity) with the human ceramide kinase (CERK [3]). Hence, it was named human ceramide kinase–like (CERKL [4]). Database searches also revealed two partial murine sequences, which, upon assembly and conceptual translation, produced a protein that was highly homologous to human CERKL (75% identity; 85.6% similarity). The human CERKL coding exons were sequenced in all members of the RP26 family. Remarkably, all patients were homozygous for a nonsense mutation (R257X; CGAÕTGA) in exon 5, which prematurely truncated the protein within the predicted kinase catalytic domain. RT-PCR analysis revealed that human CERKL is expressed in the retina, among other adult and fetal tissues. In situ hybridization on mouse eye sections showed expression of the mouse CERKL ortholog in the retina ganglion cell layer, and a faint signal was also detected in the inner nuclear and photoreceptor cell layers.
Nowadays, it is widely accepted that sphingolipid (SL) metabolism plays a fundamental role in the regulation of cell proliferation, differentiation, and apoptosis. Ceramide is at the core of sphingolipid metabolism and is the precursor of complex and bioactive sphingolipids (5). Both the central position of ceramide in SL metabolism and the increasing evidence of its involvement apoptosis highlight the significance of a finely tuned regulation of ceramide levels. Therefore, the enzymes involved in the production and clearance of ceramide must be precisely regulated. Our results suggest that CERKL may play a role in the survival of photoreceptors and other retinal cells. Moreover, recent data support ceramide-1-phosphate as a novel regulator of cell functions (6–8). Within this context, the observation that ceramide-1-phospate prevents the accumulation of ceramide by inhibiting acid sphingomyelinase in macrophages, thus avoiding apoptosis (8), is highly suggestive.
Our findings are the first genetic report linking retinal neurodegeneration in RP and ceramide-induced apoptosis, highlight SL metabolism enzymes as functional candidates for retinal disorders and establish novel targets for therapeutic intervention of these diseases.
References
1. Hims MM, Daiger SP, Inglehearn, CF. Retinitis pigmentosa: genes, proteins and prospects. Dev Ophthalmol. 2003;109-125.
2. Bayés M, Goldaracena B, Martínez-Mir A, Iragui-Madoz MI, Solans T, Chivelet P, Bussaglia E, Ramos-Arroyo MA, Baiget M, Vilageliu L, Balcells S, Gonzàlez-Duarte R, Grinberg D. A new autosomal recessive retinitis pigmentosa locus maps on chromosome 2q31-q33. J Med Genet. 1998;2:141-145.
3. Sugiura M, Kono K, Liu H, Shimizugawa T, Minekura H, Spiegel S, Kohama, T. Ceramide kinase, a novel lipid kinase. Molecular cloning and functional characterization. J Biol Chem. 2002;26:23294-23300.
4. Tuson M, Marfany G, Gonzàlez-Duarte R. Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26). Am J Hum Genet. 2004;1:128-138.
5. Hannun YA, Obeid LM. The Ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J Biol Chem. 2002;29:25847-25850.
6. Pettus BJ, Bielawska A, Spiegel S, Roddy P, Hannun YA, Chalfant, CE. Ceramide kinase mediates cytokine- and calcium ionophore-induced arachidonic acid release. J Biol Chem. 2003;40:38206-38213.
7. Pettus BJ, Bielawska A, Subramanian P, Wijesinghe DS, Maceyka M, Leslie CC, Evans JH, Freiberg J, Roddy P, Hannun YA, Chalfant CE. Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2. J Biol Chem. 2004;12:11320-11326.
8. Gómez-Muñoz A, Kong JY, Salh B, Steinbrecher UP. Ceramide-1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages. J Lipid Res. 2004;1:99-105.

Sphingolipid synthesis as a target for anti-T. Gondii drugs.

Sonda S. ¹, 2 and Pieters J. ^2
1Institut für Parasitologie, Winterthurerstrasse 266a, 8057 Zürich, Switzerland Phone: +41-1-635-8514 Fax: +41-1-635-8907 e-mail: sabrina.sonda@vetparas.unizh.ch
²Department of Biochemistry, Biozentrum der Universitaet Basel, 4056 Basel, Switzerland

Sphingolipids are ubiquitous components of plasma membranes, where they contribute to the functional heterogeneity of the lipid bilayer. In addition, sphingolipids act as second messengers in important signaling systems that regulate a wide range of cellular functions, including growth and cell cycle, apoptosis and differentiation.
Inositol phosphorylceramide (IPC) is a sphingolipid present in plants, yeast, fungi and some protozoa, but is absent in mammalian cells. The synthesis of IPC is an essential process in fungi, as inhibition of IPC synthase causes arrest of cell cycle progression followed by loss of viability.
Based on the fact that T. gondii contains an apicoplast, a plant-derived organelle that is involved in lipid synthesis, we tested the effect of Aureobasidin A, a pharmacological inhibitor of IPC synthase, on the parasite. We discovered that Aureobasidin A treatment exerts a significant dose-dependent anti-proliferative effect on T. gondii, without affecting host cell viability. Moreover the presence of the compound inhibits host cell invasion by the parasite.
We hypothesize that characterization of the IPC biosynthetic pathways present in T. gondii will (1) provide the first indication of a co-existence of plant-type and animal-type sphingolipid pathways in T. gondii, and (2) provide a potential new target for developing drugs specifically affecting T. gondii with low toxicity for humans.

Fabry disease: molecular studies in italian patients and x-inactivation analysis in female carriers.

A. Morrone¹, C. Cavicchi¹, L. Carraresi¹, T. Bardelli¹, M.A. Donati¹, S. Funghini¹, D. Antuzzi², A. Frustaci², R. Parini³, M. Di Rocco⁴, S. Feriozzi⁵, O. Gabrielli⁶, R. Barone⁷, G. Pistone⁸, C. Spisni⁹, R. Ricci², E. Zammarchi¹
1Dip. di Pediatria, Firenze; email:malmetab@unifi.it, fax 055570380, tel. 0555662543; 2Univ. Cattolica, Roma; 3Osp. ICP Milano; 4Ist. Gaslini, Genova; 5Osp. Belcolle, Viterbo; 6Clin. Ped., Ancona; 7Dip. Ped., Catania, 8Dip. di Dermatologia, Palermo; 9Un. Nefrologia, Chieti

Anderson-Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid metabolism. This multisystemic disease is caused by a primary deficiency of a-Galactosidase A enzyme (GAL A). The enzymatic GAL A defect leads to accumulation of neutral glycosphingolipids, with terminal linked galactosyl moieties, primarily in vascular endothelium and in tissue lysosomes (kidney cells, neurons, cardiomyocytes).
Fabry disease can be classified in two clinical phenotypes: the classic and the cardiac form. The former is mainly characterized, in affected hemizygous males, by angiokeratoma, acroparesthesias, hypohidrosis, pains, fever crises involvement of kidneys, brain and heart and sometimes, as the disease evolves, psychological manifestations with personality disturbances. The latter is characterized by late onset cardiomyopathy and conduction defects. Fabry patients with classic phenotype show a marked reduction in GAL A enzymatic activity, while patients with cardiac variant show residual enzymatic activity. A prevalence of Fabry disease in a referential population of male patients with a clinical diagnosis of late onset Hypertrophic Cardiomyopaty (HCM) has also been reported. Female carriers can be asymptomatic or clinically affected, usually with the mild and late onset form of the disease. They show corneal abnormalities as the most frequent clinical manifestations. Up to now more than 300 mutations have been identified in the GLA gene, mapped on Xq22. Recently, a model of the GAL A enzyme was constructed and 245 genetic lesions were plotted. This structural model could be helpful to predict, from the genetic mutation, the phenotype of the disease.
With the aim of a genotype/phenotype correlation and for better patient care and therapeutic management molecular studies were carried out on 24 Italian Fabry patients. Six new (c126-127insCATG, L167P, c617-618delTT, Q279K, c946delG, de novo A352D,) and eleven known (P40L, c124-125delAT, S78X, C174Y, IVS3+1G>A, N215S, R220X, R227Q, W236C, de novo Y365X, C378Y) mutations were identified in the genomic DNA and/or in total RNA of patients’ GLA gene. An aberrant GLA transcript c486-547del62bp, that leads to an early stop codon, was detected in a male patient carrying the IVS3+1G>A splicing defect. These results confirm molecular heterogeneity also in Italian Fabry patients.
We also detected GLA gene mutations in 26 females from 19 unrelated families with family history of Fabry disease. Mutation analysis is the only certain method of detecting female carrier status. The diagnosis in male patients can easily be made by enzimatic GAL A assay, while heterozygous females can show normal activity because of X-chromosome inactivation.
Random X-inactivation has been reported for the GLA gene. X-inactivation studies in two carrier female monozygotic twins showing a discordant expression of the same mutation of GLA gene were also described. Up to now no further studies on X-inactivation have been carried out on manifesting Fabry carriers. In order to determine an eventual correlation with clinical manifestations in Fabry carriers, X-inactivation studies were carried out on peripheral blood DNA of 26 Fabry carriers. Eleven females showed a random X-inactivation pattern and fifteen showed a moderately skewed and/or skewed X-inactivation pattern. Nine manifesting carriers showed a skewed pattern in favour of the mutant allele, while five asymptomatic carriers showed a moderate and/or skewed pattern in favour of the wild type allele. In these carriers the X-inactivation studies performed suggest a correlation between clinical manifestations and the skewing of X-inactivation. Such studies could be helpful to predict female phenotypes and give useful indications for therapeutic management.
Supported by TKT Europe 5S and Genzyme Corporation

Sphingosine 1-phosphate inhibits cell motility of C2C12 mouse myoblasts. A role for S1P2 receptor.

Becciolini L, Donati C, Nuti F, Farnararo M, Meacci E and Bruni P.
Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze,Viale G.B. Morgagni 50, 50134 Firenze; Istituto Interuniversitario di Miologia (IIM); tel ++39055413765; fax ++390554222725

Migration of skeletal muscle precursor cells is a crucial step in skeletal muscle development and post-lesional muscle regeneration. A number of growth factors including insulin-like growth factor-1 (IGF-1), hepatocyte growth factor and platelet derived growth factor have been shown to regulate positively migration of C2C12 mouse myoblasts (1). Recent studies have demonstrated that sphingosine 1-phosphate (S1P), a lipid mediator capable to exert pleiotropic actions in a variety of cell systems, is a quite unique regulator of cell motility, in that it acts as chemoattractant or chemorepellant, depending on the cell type (2). C2C12 myoblasts bear at least three different S1P receptor subtypes, namely S1P1, S1P2, S1P3 (3) and a number of different signalling pathways are initiated by the bioactive lipid in these cells (4). Here we have investigated the effect of S1P on the cell motility of these cells.
For the experiments C2C12 mouse myoblasts were routinely maintained in DMEM supplemented with 10% fetal calf serum in an atmosphere of 95% air 5% CO2. Cell migration was measured in modified Boyden chamber using polycarbonate filters, 8mm pores coated with Matrigel (4). Cells were harvested by trypsinization, washed with serum-free DMEM and were added to the upper wells at 2×104 cells per well. In some experiments cells were transfected with antisense oligodeoxynucleotides (ODN) to individual S1P receptors (5) or with pcDNA plasmid encoding for S1P2 or short interfering RNA to silence S1P2. Agonists were added to the bottom wells of the Boyden chambers. After 2 h at 37°C the filters were fixed with methanol and successively stained with Diff-Quik. The non-migratory cells on the upper membrane surface were removed and the cells which traversed and spread on the lower surface were enumerated using a Nikon microscope.
The bioactive lipid (10 nM-1 mM) dose-dependently inhibited random cell motility. Moreover, S1P (1 mM ) exerted a powerful inhibitory effect on the chemotactic response evoked by IGF-1 (10 ng/ml). The inhibition of random motility and of the IGF-1-chemotactic response by S1P resulted to be significantly attenuated in cells loaded with antisense ODN designed to inhibit S1P2 receptor. Interestingly, S1P-induced inhibition of cell motility was appreciably enhanced in S1P2-overexpressing myoblasts. Pertussis toxin treatment (200 ng/ml, 16 h) diminished random cell motility in unstimulated myoblasts but did not affect the S1P-induced biological response, suggesting that the inhibitory effect of S1P does not involve Gi-coupled receptor. These findings are in favour of a major role of signalling downstream to S1P2 receptor in the inhibition of skeletal myoblast motility induced by S1P.

References
Chargé SBP and Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol. Rev. 2004; 84: 209-238.
Takuwa Y. Subtype-specific differential regulation of Rho family G proteins and cell migration by the Edg family sphingosine-1-phosphate receptors. Biochim Biophys Acta. 2002; 1582:112-120.
Meacci E, Vasta V, Donati C, Farnararo M, Bruni P. Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C. FEBS Lett. 1999; 457: 184-188.
Okamoto H, Takuwa N, Yokomizo T, Sugimoto N, Sakurada S, Shigematsu H, Takuwa Y. Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein-coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3.

Mol Cell Biol. 2000; 20: 9247-61.
Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi Orlandini S, Francini F, Bruni P. Sphingosine 1-phosphate evokes calcium signals in C2C12 myoblasts via Edg3 and Edg5 receptors. Biochem J. 2002; 362: 349-357

Sphingosine 1-phosphate metabolism and fate in cells from the nervous system.

Riboni L, Anelli V, Bassi R, Giussani P, Viani P and Tettamanti G
Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, via Fratelli Cervi, 93, LITA-Segrate, 20090 Segrate, Milan, Italy. E-mail, laura.riboni@unimi.it; fax, +39-02-50330365; phone: +39-02-50330357.

Sphingosine-1-phosphate (S1P) has long been known as an intermediate of sphingolipid catabolism. In cells, it originates by phosphorylation of sphingosine catalyzed by sphingosine kinase and can be degraded by S1P lyase. It can also be dephosphorylated back to sphingosine by S1P phosphatase. In different cells, including cells from the nervous system, S1P has emerged as important regulator of different cellular processes including proliferation, shape and apoptosis. This sphingoid molecule is able to act both as intracellular and intercellular messenger, and different membrane receptors for it are expressed in neurons and astrocytes. Notwithstanding this, little is known on S1P metabolism and turnover in cells of the nervous system and in particular on its origin and fate in the extracellular compartment.
In this study we investigated S1P metabolism in different cells from the nervous system, including glial, neurons and brain-derived endothelial cells. To this purpose we first performed experiments by feeding cells with radiolabeled sphingosine at different doses (nM-µM). After short time pulse, 3H-sphingosine was rapidly internalized into cells in a similar fashion in all cell types; radioactive S1P was rapidly produced. The amount of 3H-sphingosine that was submitted to phosphorylation varied among cell types and increased with sphingosine doses. In all cells, 3H-S1P was submitted to degradation, detected as tritiated water in the medium. This process represented the major fate of the newly synthesized S1P in both glial and endothelial cells. In these conditions, cell medium analysis revealed the presence of extracellular S1P in all cell types. No sphingosine kinase activity was detectable in the medium, supporting that the different cells can release S1P in the extracellular compartment. In different cells, growth factors and phorbol esters stimulated sphingosine phosphorylation, without affecting its degradation. In parallel, an increase of extracellular S1P was also observed. The administration of the S1P lyase inhibitor 4-deoxypyridoxine caused a marked reduction of S1P degradation, paralleled by an increase of cellular S1P. However, this inhibitor did not affect the levels of extracellular S1P. Within the different cell types, a brain-derived endothelial cell line was found the most efficient in S1P secretion, suggesting a possible role for these cells in contributing to the extracellular S1P levels in the nervous system.
In further experiments, aiming to evaluate the turnover of extracellular S1P, 3H-S1P (50nM-5µM) was administered to cells for different times up to 1h. At all concentrations, 3H-S1P was efficiently taken up in a time-dependent fashion and about 40% of the administered radioactivity was found incorporated into cells at 1h pulse. However, intracellular 3H-S1P represented only a minor portion of the cell-associated radioactivity, the majority being associated to N-acylated sphingolipids (prevailing at lower doses), and tritiated water (prevailing at higher doses). In the presence of deoxypyridoxine, no difference in the uptake of S1P was detectable even if a marked decrease in radioactive water was observed. In this condition cellular S1P accumulated, accounting for about 50% of total uptake.
Collectively, our results demonstrate that cells from the nervous system are able to: a) regulate their S1P levels in a rapid and efficient manner; different enzymes characterized by different capacity and localization appear to be involved; to b) release in (and take up from) the extracellular compartment S1P; their metabolic equipment is also implicated in the rapid turnover of extracellular S1P.
Grant support: Italian Ministry of University, Scientific and Technological Research PRIN 2002 to L.R.

Sphingolipid metabolism in mitochondria and MAM purified from rat liver.

¹Ardail D, ²Popa I, ¹Bionda C, ²Portoukalian J
¹Laboratory of Radiobiology, Lyon-Sud Medical School, ²Department of Dermatology, Edouard Herriot Hospital, 69437 Lyon Cx 03, France (Portoukalian J, phone +33-4-72110607; fax +33-4-72110290; e-mail : portoukalian@lyon.inserm.fr)

GSLs biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER) which is transported by controversial mechanisms to the Golgi apparatus where stepwise addition of monosaccharides onto ceramides takes place. We have reported the presence of GSLs biosynthetic enzymes in a subcompartment of the ER previously characterized and termed as “mitochondria-associated-membrane” (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide-glucosyl transferase (Cer-Glc-T), glucosyl-ceramide-galactosyl transferase (Gal-T1) and sialyl transferase activities in the MAM. These enzymatic activities were found to represent at least 65% of the activities recovered in the purified Golgi apparatus, although the cross-contamination of MAM by Golgi membranes did not exceed 7.5%. Sialyl transferases activities in MAM led to the synthesis of GM3 ganglioside and small amounts of GD3.. We show that purified mitochondria as well as MAM are able to generate ceramide in vitro through both ceramide synthase or reverse ceramidase, whereas the latter enzyme activity is barely detectable in microsomes. Moreover, ceramide synthase activities were recovered in outer mitochondrial membranes as well as in inner mitochondrial membranes. Using radiolabeled sphingosine as a substrate, mitochondria could generate ceramide and phytoceramide. However, the in vitro sensitivity of ceramide synthase toward fumonisin B1 (FB1) in mitochondria as well as in MAM was found to depend upon the sphingoid base : whereas dihydrosphingosine-N-acyltransferase was inhibited by FB1 in a concentration-dependent manner, FB1 actually activated the ceramide synthase when using sphingosine as a substrate. Acylation of sphingosine-1-phosphate and dihydrosphingosine-1-phosphate generating ceramide-1-phosphate was also shown with both subcellular fractions. Moreover, the same difference in sensivity toward FB1 for the ceramide synthase activities was seen between the two phosphorylated sphingoid bases, raising the possibility that distinct base-specific enzymes may be involved as ceramide synthases.

Sphingosine-1-phosphate and liver fibrosis: friend or foe?

Sophie Lotersztajn
INSERM U581, Hôpital Henri Mondor, 94010 Créteil, France.
sophie.lotersztajn@im3.inserm.fr.

Liver fibrosis is the common response to chronic liver injury, and is characterized by increased deposition and altered composition of extracellular matrix. Its progression leads tocirrhosis and its complications (portal hypertension, hepatic failure and hepatocarcinoma), responsible for a high morbidity and mortality. The fibrogenic process is consecutive to migration of the cells to the lesions, intense proliferation and hepatic accumulation of myofibroblasts that synthetize fibrosis components and inhibitors of matrix degradation. These cells also contribute to the development of portal hypertension, due to their contractile properties. We have recently focussed on the role of S1P on the liver fibrogenesis, and used a model of cultured human hepatic myofibroblasts, that we obtain by outgrowth of explants prepared from surgical specimens of normal human liver.
We have identified S1P1, S1P2 and S1P3 mRNAs in human hepatic myofbroblasts. These receptors are functional and coupled to pertussis toxin (PTX)-sensitive and insensitive G proteins, as demonstrated in GTPgS binding assays. S1P exhibited pro and antifibrogenic properties of human hepatic myofibroblasts.
Antifibrogenic properties of S1P included inhibition of proliferation, migration and of procollagen I mRNA expression, as well as induction of apoptosis. These effects were reproduced by dihydro S1P, indicating a receptor-dependent mechanism. Analysis of the pathways involved revealed that inhibitory effects of S1P on proliferation and migration by S1P were mediated by PGE2 derived from cyclooxygenase-2 (COX-2). Indeed, S1P potently induced COX-2 protein expression. Blocking COX-2 by NS-398, a selective COX-2 inhibitor, blunted both the inhibition of proliferation and of migration by S1P, but did not affect procollagen I mRNA expression. In addition, S1P induced apoptosis of human hepatic myofibroblasts, but this occurred via a receptor-independent mechanism, since dihydro-S1P did not affect hepatic myofibroblast viability.
In contrast, S1P elicited profibrogenic properties, since the sphingolipid stimulated a receptor-dependent survival pathway. This antiapoptotic effect of S1P was PTX-sensitive and was mediated by simultaneous, but independent activations of ERK and of PI3Kinase/Akt. In addition, activation of S1P receptors triggered PTX-dependent contraction of human hepatic fibroblasts, that followed increase in intracellular calcium.
In conclusion, our results identify S1P as a novel regulator of liver fibrogenesis. The sphingolipid shows both anti and profibrogenic properties. On the one hand, S1P is antifibrogenic since it blunts proliferation, migration and procollagen I mRNA expression via a receptor-dependent pathway, and is proapoptotic via a receptor-independent mechanism. However, S1P is also profibrogenic, by promoting cell survival via a receptor-dependent mechanism. Finally, the contractile properties of S1P suggest that the sphingolipid, by regulating sinusoidal tone, may contribute to the pathogenesis portal hypertension during chronic liver diseases. Elucidation of the S1P receptors that mediate the pro and the antifibrogenic properties of S1P is underway.

Role of sphingosine 1-phosphate on NADPH oxidase activation and redox regulation of c-Src in NIH3T3 fibroblasts.

Catarzi S., Biagioni C, Favilli F., Giannoni E., Iantomasi T. and Vincenzini M.T.
Department of Biochemical Sciences, University of Florence, Viale Morgagni 50, 50134 Florence, Italy. M.T. Vincenzini: Fax 055/4222725; Tel.055/416686; e-mail: vincenzini@unifi.it.

Sphingosine 1-phosphate (S1P) belongs to groups of platelet-derived lipid mediators that regulate various biological effects. Extracellular S1P can act through specific G-protein coupled receptors (S1PRs) of the endothelial differentiation gene (EDG) family, in various types of cells including NIH3T3 fibroblasts. In these cells, intracellular S1P is also related to PDGF mitogenic signalling as second messenger and PDGF-induced cell motility by autocrin mechanism of S1PRs stimulation (1). Our previous data demonstrated that S1P and H2O2 production induced by PDGF stimulation are related to tyrosine autophosphorylation of PDGFr (2). Indeed, reactive oxygen species (ROS) such as O2-and H2O2 are signal mediators of various factors including cytokines and growth factors. A multicomponent NADPH oxidase is the enzyme mainly involved in production of ROS (3) and it is widely studied in phagocyte cells, whereas few data on activation mechanisms of this enzyme in non-phagocyte cells are reported.
The results of this study demonstrate a time dependent increase of H2O2 content in NIH3T3 fibroblasts after S1P stimulation measured by a fluorescence-based assay with DCF-DA as compared with non-stimulated cells. S1P-induced H2O2 production decreases remarkable in cells pretreated by DPI or AEBSF, specific inhibitors of flavine oxidase and NADPH oxidase respectively, indicating the involvement of this enzyme. Subsequently, we demonstrated using specific inhibitors that also phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC) activities, enzymes involved in S1P signalling, are related to the activation mechanisms of NADPH oxidase due to S1P stimulation. Indeed, both PI3K and PKC can contribute to the translocation and assembly of various components of NADPH oxidase in phagocyte cells stimulated by cytokines or other factors, but to date there are not data in non-phagocyte cells after S1P stimulation. Experiments with pertussis toxin, which inactivates Gi-coupled S1PRs, showed that S1P induces H2O2 production through the stimulation of these receptors.
Considering that c-Src, non-receptor tyrosine kinase, is related to both PDGFr and S1PRs signalling (4, 5) and is involved in signal events stimulated by ROS (6), we studied the effect of H2O2 production due to S1P stimulation on c-Src activation, in order to clarify the relationship previously found among S1P, H2O2 production and PDGF-induced tyrosine phosphorylation.
First results obtained by catalase treatment demonstrate in NIH 3T3 overexpressing c-Src that S1P-induced H2O2 generation in the presence of vanadate (inhibitor of tyrosine phosphatase) contributes to full c-Src phosphorylation and activation as compared with vanadate treated cells alone. c-Src activation is detected by immunoblotting with specific antibody of c-Src activated form that is tyr-416- phosphorylated. The treatment with inhibitors of NADPH oxidase (DPI and AEBSF) decreases c-Src autophosphorylation, suggesting the involvement of this enzyme in S1P-induced c-Src activation. To confirm a possible redox regulation of c-Src kinase activity, we exposed NIH3T3 overexpressing c-Src to 0.5 mM H2O2 for 30 min in the presence of vanadate. The results obtained show under these conditions the phosphorylation and activation of c-Src. Furthermore, no inhibition of tyr 416-phosphorylated c-Src was observed after treatment with the inhibitors of NADPH oxidase, PI3K and PKC activity indicating that S1P activation of these enzymes is an upstream event of c-Src activation. These data demonstrate that S1P and its receptors are involved in NADPH oxidase activation and redox regulation of c-Src kinase activation and these events can be important factors for the cross communication between receptor tyrosine kinase and G-protein coupled receptors.
References
1. Hobson JP, Rosenfeldt HM, Barak LS, Olivera A, Poulton S, Caron MG, Milstien S, Spiegel S. Role of the sphingosine-1-phosphate receptor EDG-1 in PDGF-induced cell motility. Science. 2001; 291:1800-1803.
2. Catarzi S, Iantomasi T, Biagioni C, Favilli F, Vincenzini MT Sphingosine-1-phosphate receptor involvement on the tyrosine phosphorylation of PDGF receptor in NIH3T3 fibroblasts. Fifty European Symposium of the protein society2003, Florence 29 Marzo -2 Aprile Abstract
3. Finkel T Oxidant signals and oxidative stress Curr. Opin. Cell Biol. 2003; 15, 247-254
4. Rakhit S, Conway AM, Tate R, Bower T, Pyne NJ, Pyne S. Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle. Role of endothelial differentiation gene 1, c-Src tyrosine kinase and phosphoinositide 3-kinase. Biochem J. 1999; 338:643-649
5. Conway AM, Rakhit S, Pyne S, Pyne Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase. Biochem J. 1999; 337:171-177.
6. Yoshizumi M, Abe J, Haendeler J, Huang Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species. J Biol Chem. 2000; 275:11706-11712.
This work was supported by grants from MIUR and by a donation from the Cassa di Risparmio di Firenze

Characterization of the ceramide moieties of sphingoglycolipids containing sphingadienine fromhuman brain with Alzheimer’s disease by ESI-MS/MS.

Colsch B ¹,2, Afonso C ²,Fournier F ², Portoukalian J ³, Tabet JC ², Baumann N 1.
¹INSERM U495, Paris, France; ²CNRS UMR 7613, Paris, France ;³INSERM U346, Lyon, France.
Mailing address: Nicole Baumann INSERM U 495, Salpetriere Hospital, 75651 Paris cedex 13; Fax: 33 1 4584 8008; Tel: 33 1 4216 2155 ; e-mail: baumann@ccr.jussieu.fr

We have shown previously by ESI tandem mass spectrometry, that adult mouse brain contains ceramides from galactocerebrosides and sulfatides with both sphingosine and sphingadienine [1]. In this study we were interested in using a similar methodology for a comparative analysis of sphingoglycolipids present in humain brain with Alzheimer’s disease.
Lipids extracts were obtained by chloroform-methanol extraction, and Folch partition from human brain. The upper phase containing the gangliosides and a sulfatide fraction were further purified on C18-silicic acid cartridges followed by RP-HPLC separation. The lower phase containing galactocerebrosides and free ceramides were purified using LC-NH2 cartridges. Control of the fractions purity was carried out by HP-TLC. Mass spectrometry characterization was performed by electrospray ionization using both negative and positive mode. An ESI-ion trap mass spectrometer (Bruker Esquire 3000) was used in MSn in order to characterize ceramide structures. A triple quadrupole instrument (Micromass Quattro) was employed in parent and neutral loss scan mode to obtain additional information.
By using the same methology that was used in our previous study on mouse brain [1], we have found that sphingadienine is present in human brain ceramide moieties of the sphingoglycolipids. The relative fatty acid composition is different than what is observed in the mouse with a lower proportion of short chain fatty acids [C16:0 to C22:0 (OH)] and a greater proportion of very long chain fatty acids [C24:1 to C26:0 (OH)]. In Alzheimer’s disease, a reduction of the most abundant sulfatide species and an increase of ceramides in the total lipid extract has been observed [2]. After purification of the isoforms of sulfatides, galactocerebrosides and ceramides, we have confirmed that the sulfatide abundance was decreased. It concerns both the sulfatides with sphingosine and those with sphingadienine. We have also observed an increase of various ceramide isoforms.
References[1] Colsch B, Afonso C, Popa I, Portoukalian J, Fournier F, Tabet JC, Baumann N. Characterization of ceramide moieties of sphingoglycolipids from mouse brain by electrospray-tandem mass spectrometry : identification of ceramides containing sphingadienine. J Lipid Res. 2004, 45: 281-286[2] Han X, Holtzman D, McKeel D, Kelley J, Morris J. Substantial sulfatide deficiency andceramide elevation in very early Alzheimer’s disease : potential role in disease pathogenesis. J Neurochem 2002, 82 : 809-818.

Cytosolic sialidase Neu2 upregulation during PC12 cells differentiation.

F. Colombo, A. Fanzani, R. Giuliani, M. Bresciani, A. Preti, and S. Marchesini
Unit of Biochemistry, Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy.

The cytosolic sialidase Neu2 is known to be involved in myoblast differentiation (1,2). Despite its relative abundance in skeletal muscle, cytosolic sialidase was found also in other tissues, such as liver (3), thymus (4) and, although at low levels, brain (5).
RT-PCR experiments and “gene reporter” analysis of a 1.4 Kb fragment of Neu2 rat promoter were performed to obtain the expression levels of Neu2 sialidase in PC12 cells, a favored model to study neuronal differentiation. As a consequence, a Neu2 transcriptional induction during nerve growth factor (NGF), fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) treatments was found in this cell line, whereas the Neu2 transcript was absent in untreated cells. In addition, the expression analysis of four Neu2 deleted promoters revealed a remarkable increase of luciferase activity in NGF treated PC12 cells, suggesting that the Neu2 transcriptional level is highly regulated. Interestingly, the major increase of luciferase activity was found after deletion of a fragment containing a rat dispersed repetitive element, a genomic element able in some cases to modulate transcription and translation efficiency (6).
Enzymatic assays revealed a cytosolic sialidase activity only during the early steps of NGF differentiation at days 1-2 of treatment, and the low enzymatic activity strictly correlated with the transcript expression levels observed. As a consequence, PC12 cells stably transfected with an expression vector harboring the rat Neu2 cDNA showed a sialidase activity up to 25-times higher than the NGF induced endogenous activity. These data suggest a possible involvement of cytosolic sialidase Neu2 in differentiation of PC12 cells (7).
References
[1] Sato, K. and Miyagi, T.(1996) Bioch. Bioph. Res. Comm. 221, 826-830.[2] Fanzani, A., Giuliani, R., Colombo, F., Zizioli, D., Presta, M., Preti, A., Marchesini S. (2003) FEBS Lett. 547, 183-8.[3] Miyagi, T. and Tsuiki, S.(1985) J. Biol. Chem. 260, 6710-6.[4] Kotani, K., Kuroiwa, A., Saito, T., Matsuda, Y., Koda, T., Kijimoto-Ochiai, S.(2001) Biochem. Biophys. Res. Commun. 286, 250-8.[5] Hasegawa, T., Feijoo Carnero, C., Wada, T., Itoyama, Y., Miyagi, T.(2001) Biochem. Biophys. Res. Commun. 280, 726-32.[6] Landry, J.R., Medstrand, P., Mager, D.L. (2001) Genomics 76, 110-6.[7] Fanzani, A., Colombo, F., Giuliani, R., Preti, A., Marchesini S. (2004) FEBS Lett. 566, 178-82.

IGFBP-3 is a survival factor for human endothelial cells and promotes in vitro angiogenesis through activation of sphingosine kinase.

Riccarda Granata¹, Letizia Trovato¹, Giusy Sala², Giovanni Garbarino¹, Riccardo Ghidoni², Ezio Ghigo^1
1Dept Internal Medicine, Unversity of Turin; ²Laboratory of Biochemistry and Molecular Biology San Paolo Medical School, Milan, Italy

Insulin-like growth factor binding protein-3 (IGFBP-3), the major carrier of IGF, exerts either growth promoting or growth inhibiting effects in different cell types. We have previously found that IGFBP-3 inhibits human umbilical vein endothelial cell (HUVEC) apoptosis induced by serum starvation by decreasing the proapoptotic sphingolipid ceramide. Moreover IGFBP-3 was found able to enhance cell motility and to increase IGF-I release, suggesting a new role in endothelial cell survival and angiogenesis. Herein we show that IGFBP-3 (1000 ng/ml) promoted HUVEC survival and inhibited apoptosis in serum-free conditions by activating the enzyme sphingosine kinase (SK). SK catalyses the formation of sphingosine-1-phosphate (S1P), a lipid second messenger implicated in the stimulation of cellular proliferation and inhibition of apoptosis. N,N-dimethylsphingosine, an inhibitor of SK potently blocked IGFBP-3 survival effect, suggesting that IGFBP-3 acts through the SK signaling pathway. Furthermore, IGFBP-3 stimulated the IGF-I receptor (IGF-IR) tyrosine phosphorylation and IGFBP-3 antiapoptotic effect was blocked by the IGF-IR tyrosine kinase inhibitor tyrphostin AG1024. IGFBP-3 even activated the survival pathways PI3K/Akt and ERK which are known to be involved in both IGF-I and S1P signaling. Finally we show that IGFBP-3 dose-dependently induced in vitro formation of vessel-like tubular structures by HUVEC in growth factor-reduced Matrigel. In all, our findings demonstrate that IGFBP-3 promotes endothelial cell survival and inhibits apoptosis via the sphingolipid pathway down-regulating ceramide and activating SK to increase S1P. These effects are likely mediated by the IGF-IR signaling pathway.Enhancement of HUVEC motility and induction of vessel-like structure formation suggest a role for IGFBP-3 to promote angiogenesis.
Study supported by MURST, University of Turin and SMEM Foundation

Resveratrol impairs the 3D aggregation of MDA-MB-231 through a ceramide-mediated mechanism.

Ersilia Dolfini¹, Leda Roncoroni¹, Giusy Sala², Paola Signorelli², Elena Dogliotti¹ , Nicoletta Sacchi³, Riccardo Ghidoni^2
1Dept Biology and Genetics, Univ Milan, Milan, Italy, ²Laboratory of Biochemistry and Molecular Biology San Paolo Medical School, Milan, Italy, 3Dept Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY- riccardo.ghidoni@unimi.it

Resveratrol, a polyphenol present in grapes and wine, exerts a drastic growth-inhibitory effect on the human breast cancer MDA-MB-231 cell line grown in the 2D-culture. We recently described the mechanistic relationship between growth inhibitory/pro-apoptotic effect of resveratrol and de novo synthesis of ceramide [Scarlatti et al FASEB J (2003) 17,2339-41].
Here we report that ceramide mediates the growth inhibitory effects of resveratrol in MDA-MB-231 cells grown in three-dimensional (3D) cultures. MDA-MB-231 when grown in 3D-cultures can form multicellular spheroids (MCSs). First we treated fully grown MCSs with resveratrol (32 mM, 48 hours) and observed an accumulation of ceramide in the MCSs, in concomitance with the disruption of the most external spheroid cell layer(s) and loss of the clonogenic potential.
Second, we observed that treatment of MDA-MB-231 cells with 32 mM resveratrol significantly impaired the formation (number and size) of 3D-MCSs. This effect correlated with a time-dependent accumulation of endogenous ceramide. In addition, cells disaggregated from the few MCSs that were able to form MCSs after resveratrol treatment lost their clonogenic potential.
Thus, resveratrol can both reduce full-grown MCSs and prevent the formation of MCSs by triggering ceramide-dependent pathways. These results indicate that resveratrol-ceramide signaling is indipendent from the sovracellular culture level.

Sphingomyelin metabolism modifications in response to the effect of high neutron energy.

Perrella G.°, Cataldi S.*, Toller M°., Meli A°., Ranaldi F•, Messina C#., Viola Magni M.P.* and Albi E.*.
°Department of Experimental and Clinical Pathology and Medicine University of Udine.
*Department of Biochemical Science and Molecular Biotechnology, Physiopathology, Perugia.
•Department of Biochemical Science University of Firenze
#Dipartimento di Biologia Marina Universita di Trapani.

Classic radiation dosimetry, performed with traditional physical methods, does not evaluate the kinetics of radiation absorbance or the biological response(s) to radiation. Those are key aspects in biological systems and cells, which possess elaborate radiation response mechanisms (DNA repair, cellular components turnover, apoptosis, etc.). Biological dosimetry on eukaryotic cell cultures provides nowadays elaborate experimental targets very close to animal and human organs or organisms, which can be used as radiation target systems. In this study, we used FRTL5 cells, strain of normal and differentiated follicular thyroid cells, well characterized, presently an international standard for bioassay of autoimmune thyroid diseases. The FRTL5 cell strain permanently expresses in vitro most of the in vivo tissue-specific thyroid functions, such as for Thyrotropin (TSH) sensitivity for Thyroglobulin synthesis and secretion, Iodide active transport, Peroxidase production, etc. The aim of this study is to investigate the sphingomyelin metabolism modifications in response to neutron irradiation in under the following different physiological conditions: 1) in proliferative state, under Thyrotropin effect. 2) in non proliferative state, in TSH-deprived culture medium. In both conditions the FRTL5 cells have been exposed to neutron irradiation (558 mSv/h) in the laboratory of CERN of Gineva. Moreover for both conditions non irradiated FRTL5 cells have been used as control. The obtained results show that: 1) In the cellular homogenate, exposure of FRTL5 cells to neutron irradiation appears to strongly inhibit the neutral sphingomyelinase activity and to slowlyinhibit sphingomyelin synthase and phosphatidylcholine-dependent phospholipase C activity only in proliferative state. 2) At nuclear level, exposure of FRTL5 cells to neutron irradiation appears to increase significantly the neutral sphingomyelinase activity and to inhibit the phosphatidylcholine-dependent phospholipase C activity in non proliferative cells. No significant effects are reported for sphingomyelin-synthase. We can speculate that the different responses of the high neutron energy on the cellular and nuclear sphingomyelinase and phosphatidylcholine-dependent phospholipase C activities are due to the presence of different isoforms with different functions. In the active proliferating cells the sphingomyelinase produced in the homogenate decreases whereas in the non proliferating cells there is an increment in synthesis of sphingomyelinase in the nucleus and also an inhibition of phosphatidylcholine-dependent phospholipase C activity. In non proliferative cells the data show an incrementof nuclear ceramide and a decrement of diacylglycerol pool, probably involved in cell deth. The correct balance ofceramide and diacylglycerol in the nucleus is essential for cell viability. Alteration in this balance could be related to apoptosis.

Dexametasone reduces cell growth via nuclear sphingomyelin metabolism.

Tringali Sabina, Tringali Anna Rita, Viola Magni MP, Albi E
Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100, Perugia.

Corticosteroids have effects on the function of most cells. In the whole animal deficiency or chronic excess is incompatible with survival. Glucocorticoid hormones have been implicated as regulators of T-lymphocyte growth and differentiation e/o apoptosis. Recently studies show that dexamethasone acts via sphingomyelin (SM) metabolism. In fact, it increases SM content, regulates the SM-synthase and sphingomyelinase (SMase) activity. The biological effects of dexamethasone are generally thought to be mediated by an intracellular receptor protein which transduces the hormone signal to the nucleus and participates directly in gene regulation. Since inside the nucleus the metabolism of the SM in relation to cell function has been described, the aim of this work is to establish the possible effects of dexamethasone on cellular and nuclear SM metabolism in tumour cells. At this end, the lymphoma cells (SUP-T1) were seeded in RPMI 1640 in the presence of 10% fetal bovine serum, at a concentration of 100,000/ml and cultivated until 6 days in a 5% CO2 humid atmosphere.Two lots of 5 ml were prepared: one control and one cultivatedin the presence of dexamethasone (500mg/ml). The cell growth was monitored and theSMase and SM-synthase assayed in the homogenate and nuclei.
The results show a growth inhibition of 66% after dexamethasone treatment respect to the control cells. In the same cells an increase of homogenate SMase activity reachs a peakafter 2 hours when the SM-synthase activity is inhibited. The nuclear SMase activity show aprogressiveinhibition between 2 and 24 hours. It is possible that the dexametasone inhibits the cell growth acting not only on cellular sphingomyelin metabolism enriching early the ceramide pool but also on nuclear SM. The inhibition of nuclear SMase induces a reduction of nuclear ceramide pool and an increase of SM. Since it may be postulated that SM acts by stabilising chromatin structure, it is possible that changes in nuclear SMase activity may be involved in the regulation of cell cycle not through the action of ceramide production but through the influence of SM at the level of chromatin structure. After dexamethasone treatment, SM could contribute to favour chromatin clamps formation in apoptotic process.

Cholesterol/sphingomyelin relationships in oncohaematologic patients.

Pugliese L., Bernardini I., Viola Magni MP, Albi E.
Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100, Perugia.

Cholesterol (CHO) is a molecule widely distributed in living organisms. It is located mainly at the level of cell membranes in the unesterified form and in the blood as a component of lipoproteins mainly in the esterified form and it has a role in many physiological and pathological processes. Numerous studies have shown that a strong interaction exists between unesterified CHO and sphingomyelin (SM) which arises from the van der Waals interaction between CHO and the saturated lipid acyl chains. The cellular concentrations of SM and CHO are positively correlated in several pathological and experimental conditions. The CHO-SM relation suggests a mechanism for sterol distribution in which sterol levels are determined by the intrinsic characteristics of each membrane. Epidemiologic studies have indicated a relation between lipids and cancer and abnormal blood lipid profiles have been reported in human malignancies. Statistically significant values of CHO were reported in oncohaematologic patients. A significant increase in CHO levels was observed in all patients responding to therapy. The aim of this work was to highlight if the low CHO levels in patients with Monoclonal Gammopathy are accompanied by modification in the SM level. Blood samples of hospitalized patients with Monoclonal Gammopathy with low CHO level (Experimental) were collected. Patients with Monoclonal Gammopathy with a normal CHO level were used as control (Control) . Serum samples were stored at -20°C until used. The level of CHO and SM was measured in untreated and sphingomyelinase treated samples. The results show that the serum CHO and SM levels in experimental patients are 55% and 61% compared to control. After digestion of SM with SMase in both group of patients a decrease in CHO and an increase in CHO esters, was observed. The effect of SMase on SM hydrolysis was low. After three washings of the inorganic phase with chloroform/methanol and solvent containing NaCl, an increase of CHO and CHO was reported; the values were higher in experimental samples.It is possible that in the patients with Monoclonal Gammopathy there is a metabolic and structural modification of the sphingomyelin which forms a pathological complex with CHO influencing the effect of the therapy.

Lipid rafts in hepatocyte nuclei.

Villani M.*, Cascianelli G.*, Tosti M.#, Viola Magni MP.*, Albi E.*
*Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100 Perugia, Italy. e-mail: ispatgen@unipg.itealbi@unipg.it
#Dipartimento scienze biopatologiche veterinarie, Università di Perugia.

Lipid composition has been described as being responsible for the presence of domains in the membrane. The main role in the process of domain formation is played by cholesterol (CHO) and sphingolipids.These cholesterol-enriched domains may be stabilised by the presence of saturated alkyl chains of sphingomyelin. Recent biophysical data have provided clues to show that changes in membrane structure are a critical function. Lipid raft microdomains were conceived as part of a mechanism for intracellular trafficking of lipids and lipid-anchored proteins.The composition and function of membrane rafts can be modulated in response to a variety of factors and stress conditions. In fact the membrane rafts are the specific sites for ceramide generation in response to various agonists. Aida et al. (2002, 1) suggest that sphingomyelin-cholesterol microdomains or rafts form a stable lipid matrix, which act as an ordered support for receptor-mediated signaling events. Recently we have demonstrated, inside the nucleus, that the amount of cholesterol is similar to that of sphingomyelin, and it increases in chromatin digested with exogenous sphingomyelinase or proteinaseK, suggesting the existence of a chromatin CHO-SM linked fraction (2).
Moreover, during liver regeneration, an increase in chromatin cholesterol was observed between 6 and 18 hours after hepatectomy, when the neutral-sphingomyelinase activity increases and the sphingomyelin-synthase is inhibited. In the present work the first aim was to isolate the lipid rafts from hepatocyte nuclei. After nuclei Triton X-100 treatment, isolation of lipid rafts by sucrose density gradient centrifugation was performedaccording to Danielsen (1995, 3). On isolated rafts, the lipid, sphingomyelinase activity and the electron microscope activitywere studied. The results show that the morphology of lipid rafts is similar to that previously reported for microvillar membrane and they are enriched with spherical vesicles, suggesting that these are the mechanism for intranuclear molecule transport in relation to cell function. The nuclear lipid rafts contain CHO and SM and a high activity of sphingomyelinase is detected. Since the nuclear SM, its metabolites and CHO play a role in inducing cell proliferation or apoptosis, it is possible that their function is due to microdomain organization.
1) Aida et al., (2002) FEBS Letters 531, 47-53
2) Albi and Viola Magni (2002) Journal of Hepatology 36, 395-400
3) Danielsen (1995)Biochemistry 34, 1596-1605

Effect of protein kinase C delta on nuclear sphingomyelin metabolism.

La Porta C.A.M.*, Cataldi S., Lazzarini, R, Viola Magni,MP., Albi E.
*Department of Biomolecular Science and Biotechnology, University of Milan e.mail:caterina.laporta@unimi.it; Department of Biochemical Sciences and Molecular Biotechnology Physiopathology, Policlinico Monteluce, 06100 Perugia, Italy. e-mail: ispatgen@unipg.it ealbi@unipg.it

Nuclear sphingomyelin metabolism changes during cell proliferation and apoptosis due to modifications in sphingomyelinase (SMase) and sphingomyelin-synthase (SM-synthase) activity (1). The first enzyme hydrolyses sphingomyelin (SM) to produce ceramide, and SM-synthase forms SM by using the phosphorylcholine of phosphatidylcholine (PC) so producing diacylglycerol (DAG). Until now the mechanism of SM metabolism modification in relation to cell function has not been described. Since the protein kinase C delta (PKC delta) has an inhibitory effect on SMase activity (2), in fact specific antibodies anti PKC delta increase the SMase activity significantly, and SM-synthase activation attenuates SMase-induced signalling (3), the present research was aimed at investigating the effect of PKC delta overexpression on nuclear SM metabolism in relation to the induced apoptotic process. At this end we chose B murine melanoma cells (BL6), that do not express the PKC delta and in which overexpression of this enzyme was induced, as the experimental model (4). The overexpression of such an isoform induces a slight increase of cells into apoptosis and an increase of arrested cells into G0/G1 (4). Radioactive PC and SM were used as precursors for evaluating either PC-PLC or SM-synthase and SMase respectively. Incubation of cell homogenate showed no significant modifications of PC-PLC, SMase and SM-synthase activities whereas in the nucleus inhibition of SMase and activation of SM-synthase were demonstrated in transfected cells. No modifications were found for nuclear PC-PLC activity. In chromatin, isolated from the tumoral mass, the overexpression of PKC delta determined the PC-PLC and SMase inhibition whereas the SM-synthase reached a higher level of the activity than that found in the isolated nuclei. Therefore the PC content decreased and the SM content increased. The decrease in PC was due mainly to the SM-synthase activity whereas the pathway through PC-PLC is inhibited. In conclusion, the overexpression of PKC delta, which in murine melanoma cells shifts from the cytoplasm to the nucleus in relation to cell cycle (5) determines an increase in SM content in whole nuclei, so probably modifying the nuclear membrane structure and a greater increase in chromatin SM which appears to be related to protein synthesis present in the apoptotic process.
1) Albi,E. et al.. J. 2003 Cell Physiol. 196:354-61; 2) Visnjic D et 1999 Biochem. J. 344, 921-928; 3) Hiutema, K. et al. 2004 EMBO J. 23, 33-44; 4) C.A.M. La Porta et al.2000 Melanoma Research 10:93-102; 5)Perego C., et al., 2002. Biochem Biophys Res Commun 294, 127-131.

Clinical and genetic heterogeneity in GM1 gangliosidosis patients as a result of alteration in β-galactosidase enzyme and elastin binding protein.

A Caciotti, MA Donati, A Boneh¹,  A d’Azzo², T Bardelli, S. Malvagia, V Kimonis³, R Parini⁴, D Antuzzi⁵, A Federico⁶, E Zammarchi, A Morrone.
Dept. of Pediatrics, Meyer Hospital, Florence, Italy, email:malmetab@unifi.it, fax 055570380, tel. 0555662543; Metab Serv, Victoria Australia¹, Genetics St. Jude H, Memphis, USA², Children’s Hospital, Boston, USA³, Neurology, Siena, Italy⁴, Catholic Univ, Rome, Italy⁵, Pediatrics, Monza, Italy⁶

A deficiency of the human acid b-galactosidase (GLB1) results in two lysosomal storage disorders: the sfingolipidosis GM1-gangliosidosis and the mucopolisaccharidosis Morquio B disease. The clinical heterogeneity of GM1-gangliosidosis is expressed by three major phenotypes: infantile (type I), late infantile or juvenile (type II), and adult or chronic (type III). To date several therapeutic approaches, including substrate deprivation and treatment with “chemical chaperone” molecules, have been evaluated for some lysosomal storage disorders, but most of these diseases, such as GM1-gangliosidosis and Morquio B, remain untreated.
GLB1 gene gives rise to the GLB1 lysosomal enzyme and to the elastin binding protein (EBP), whose primary deficiency is linked to impaired elastogenesis. In addition, galactosugar-bearing moieties have been demonstrated to alter EBP function and in turn to cause impaired elastogenesis. GLB1 forms an high molecular weight multienzyme complex with the glycosidases [beta galactosidase (GLB1), alfa Neuraminidase (NEU1) and N-acetylaminogalacto-6-sulfate sulfatase (GALNS)] and the protease [(lysosomal carboxypeptidase A (PPCA)] inside lysosomes, while EBP is bound to PPCA and NEU1 on cell surface.
Here we report the clinical, biochemical and genetic characterisation of 11 GM1 patients covering the entire spectrum of GM1- gangliosidosis phenotypes. Molecular analysis was carried out on the patients’ genomic DNA and total RNA isolated from cultured fibroblasts, by direct sequencing of GLB1 cDNA, genomic DNA and enzymatic restriction analysis. With exception of a juvenile patient, carrying the new C230Y and the known R201Hlocalized in a region of GLB1 pre-mRNA encoding only for the lysosomalenzyme, all other genetic lesions identified affect both GLB1 and EBP cDNA and these proteins, altered in function and distribution, contribute differently to the specific clinical manifestations. With the aim of assigning a putative role of these mutated amino acids in the GLB1 stability or catalysis, expression studies and enzyme assays were performed. These studies enabled a genotype/phenotype correlation. Sequence analyses of GLB1 related proteins between species, suggest or confirm correlation between pathological molecular and clinical findings. Expression studies on COS1 cells and Western Blot analyses showed that the identified mutations affect GLB1 enzyme activity and/or stability. In addition, some GLB1 gene mutations do not appear to be stabilized by PPCA probably because these mutations hamper the interaction between GLB1/EBP and PPCA inside multienzyme complex. Impaired elastogenesis in fibroblasts of some patients with infantile form, as detected by elastic fibers assembly by immunofluorescence studies, might be associated with a primary defect of EBP function, given that its normal amount remained unchanged. In a patient with the juvenile form, who shows large urinary keratan sulfate excretion and mutations affecting only GLB1 enzyme, a mild EBP reduction and a decreased elastin deposition were present. These data suggest that the keratan sulfate accumulation leads to secondary EBP deficiency with impaired elastogenesis.
In order to study the effect of the GSL biosynthesis inhibitors (NB-DNJ) and of the chaperone molecules (DJG and galactose), the skin fibroblasts of three patients with three different phenotypes were pharmacological supplemented with galactose and galactose inhibitors. Interestingly the GLB1 enzymatic activity assayed in the adult patient’s fibroblasts, cultured in the presence of galactose, showed a considerable increase of GLB1 activity. These results could be useful for the development of therapeutic strategies.
Supported by Azienda O-U Meyer, AMMEC and MPS Italy

Sphingolipid Metabolism and Cell Regulation

Yusuf A. Hannun
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA

Studies over the past 2 decades have underscored the importance of specific sphingolipids such as sphingosine, ceramide, and sphingosine phosphate as bioactive molecules involved in diverse cellular responses. As a consequence, many of the enzymes of sphingolipid metabolism have emerged as regulated components in signaling and cell regulatory pathways. The direct targets for these sphingolipids (such as receptors, kinases, and phosphatases, have also emerged as important signal transducers.
More recent studies have introduced another layer of complexity to the understanding of bioactive sphingolipids which derives from the compartmentalized metabolism and action of these lipids. In our studies, we find that the action of ceramide is dependent on the specific compartment of metabolism and generation. One emerging specific pathway connects de novo synthesis of ceramide to activation of the protein phosphatase PP1 and the dephosphorylation of nuclear SR proteins. This serves as a prototype “singling module”. The versatility of sphingolipid metabolism and its interconnection raise the possibility that these individual modules may allow for “integration” of cell responses. This talk will discuss these specific examples of ceramide function and the emerging concepts of sphingolipid metabolism and function.

Can the nuclear sphingomyelin regulate the fate of the cell?

Albi, E., Serenelli, G., Rossi, G., Cataldi, S., Borio, T., Lazzarini, R. Viola Magni, M.P.
Dipartimento di Scienze Biochimiche e Biotecnologie Molecolari, Sezione di Fisiopatologia, Policlinico Monteluce, Perugia

The presence chromatin phospholipid (PLs) fraction rich in sphingomyelin (SM) and of enzymes including neutral-sphingomyelinase (N-SMase, 1), SM-synthase (2) and reverse SM-synthase (3) related with SM metabolism has been shown. Since the activity of these enzymes may modify the amount of diacylglycerol (DAG) or ceramide inside the nuclei, the present research was aimed at investigating the chromatin SM metabolism in cell proliferation, differentiation and/or in apoptotic process. For the proliferation study, the regenerating liver was used as experimental model; the rats were hepatectomised between 8-10 a.m. and killed 6,12, 18, 24 hrs after operation. As control, sham operated animal were used.
For differentiation and apoptotic studies, the erytroleukemic cells treated with DMSO and/or vitamin D3 were used as experimental model. The murine virus-induced Friend cell (Clone 745A) were grown for 5 days in RPMI 1640 medium containing 10% fetal calf serum at 37°C in 5% CO2 humid atmosphere; experimental samples were cultivated as above in the presence of 1.5% DMSO or 10 mMol vitamin D3 or both . Radioactive SM was used as precursors for evaluating N-SMase and reverse SM-synthase activities and radioactive PC for SM-synthase activity. In rat liver regeneration, the activity of SMase and reverse SM-synthase increases at the beginning of S-phase when the SM-synthase is inhibited, so the chromatin ceramide pool is enriched when the cell proliferation start. The differentiated cells after 5 days of culture in the presence of DMSO are 43% and increase to 74% if vitamin D3 is added. The presence of the vitamin D3 alone induces the cells in apoptosis. The SMase and reverse SMase activities, which enriched the chromatin ceramide pool, increase during the apoptotic process whereas the SM-synthase activity, which enriched the chromatin DAG pool, increase during cell differentiation. These results may lead to the hypothesis of the existence of internal equilibrium between different messengers essential for a cell physiological life.
1) Albi,E. and Viola Magni, M.P. Biochim. Biophys. Res. Commun., 236, 29-33, 1997; 2) Albi,E., and Viola Magni,M.P. FEBS Letters 460, 369-372, 1999

Purification and characterization of a sphingomyelin synthase activity from pseudomonas aeruginosa.

Chiara Luberto, Elizabeth Collins, Samer El-Bawab, and Yusuf A. Hannun
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA

We report the purification and biochemical characterization of an enzymatic activity that synthesizes sphingomyelin (SM) in cultures of Pseudomonas aeruginosa. SM synthase-like activity was found secreted in the medium in cultures of Pseudomonas aeruginosa, strains PA01 and PAK whereas it could not be detected in cultures of Escherichia coli. Such activity showed a neutral pH optimum. From the medium of PAK cultures, SM synthase was concentrated by precipitation with 60% ammonium sulfate and found to bind to hydrophobic matrices such as butyl and octyl sepharose in the presence of high concentrations of salt. After eluting the protein from octyl sepharose columns, fractions containing peak activity were applied onto blue sepharose, mono P, and finally onto mono S columns. Fractions containing SM synthase activity were then resolved on 8 and 12% polyacrylamide-SDS gels and proteins were visualized by silver staining. In the lanes containing peak SM synthase activity three to four silver stained bands correlated with the enzymatic activity. These bands were therefore cut and sent for peptide sequencing. From the mono P column the isoelectric point of the protein was calculated to be approximately 4.9. Moreover, using a superose 12 gel filtration column the molecular weight was estimated to be approximately 60 to 70 kD. Interestingly, a sphingomyelin hydrolysing activity co-purified with SM synthase throughout the purification protocol. Using the fractions with peak activity obtained from the mono S column it was shown that SM synthesis was detected only when phosphatidylcholine or lysophosphatidylcholine and not phosphorylcholine or CDP-choline were used as substrates. Phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol were not used as substrates in the presence of NBD-C6-ceramide by the purified transferase activity.

Glycospingolipid antigens involved in cd1 mediated T-cell response: synthesis and preliminary bio-evaluation.

Federica Compostella(a) Laura Franchini(a) Luigi Panza(b) and Fiamma Ronchetti(b)
(a) Dipartimento di Chimica e Biochimica Medica, Università di Milano
(b) Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Università del Piemonte Orientale, Novara

CD1 proteins are a family of antigen presenting molecules strictly related in structure and evolutionary origin to the major histocompatibility complex (MHC) encoded molecules. However, in contrast to the MHC encoded products, CD1 molecules restrict T-cells response to non-peptide lipid and glycolipid antigens.
In particular, it has been demonstrated that sulfatide, a mixture of galactosylceramides with different fatty acids bound to the amino group of sphingosine and sulfated in position 3, is the antigen presented by a CD1a protein.
We are involved in a project aimed at elucidating the antigen structural features required for the recognition by CD1, and for T-cells activation; in this context we decided to investigate the influence of variations in the sulfatide lipid tails on the recognition of CD1a-restricted T-cells; thus, we planned the synthesis of various sulfatides, differing in the acyl chain length, which are represented in the natural mixture (Fig. 1).

In the present communication we will describe the synthesis and a preliminary bio-evaluation of such a family of sulfatides.
In order to obtain the sulfatide by synthesis, it is necessary to have access to the aglycone part, the ceramide. In particular, we focused our attention to 3-OH protected azidosphingosine as it is well known that it is a better substrate than ceramide in glycosylation reactions. New syntheses of azidosphingosine starting from chiral pool have been realized. The so obtained sphingosine derivative has been glycosylated with a galactosyl donor and different fatty acids have been introduced. Final sulfation at position 3 of the galactose moiety allowed to obtain the desired sulfatides.
The synthetic compounds have been tested for activation of sulfatide-specific T cells and compared to a commercial sample of sulfatide isolated from bovine brain and containing the natural fatty acids mixture on ceramide moiety. The activity of the synthetic and natural sulfatides was studied through the release of TNF-alpha or IL-4 as T cell activation read out. The results showed that the analyzed T cells are differently activated by the synthetic sulfatides with respect to the natural mixture.

Prion protein-ganglioside association in microdomains of lymphocyte plasma membrane.

V. Mattei, M. Sorice, *A. Pavan
Dip. Medicina Sperimentale e Patologia, Università “La Sapienza”, Roma
*Dip. Medicina Sperimentale, Università di L’Aquila

Prionic protein may be cause a series of fatal encephalopathies such as Creuzfeldt-Jakob disease of humans, scrapie of sheep and bovine spongiform encephalopathy. All protein diseases are characterized by the conversion of the constitutive cellular prion protein (PrPC) into the pathogenic form, which is also known as scrapie PrP (PrPSc). Recent evidences showed that PrPC has been detected in human lymphocytes, where the level of PrPC was regulated as a consequence of T cell activation. Since PrpC is bound to the cell surface by glycosylphosphatidylinositol (GPI)-anchored linkage, in this study we evaluated the presence of PrPC in microdomains, which are specialized glycosphingolipid-enriched plasma membrane domains, involved in signal transduction pathway. We preliminarly analyzed protein distribution on the plasma membrane of both lymphocytic and neuronal cells. The results, obtained by scanning confocal microscopy, revealed the presence of large clusters. In order to clarify whether these clusters are corresponding to microdomains, we analyzed PrPC association with specific gangliosides. The presence of PrPC in microdomains was further confirmed by their presence in the Triton-insoluble fractions, obtained by sucrose linear gradient. Additional evidence for a specific PrPC-ganglioside interaction in these cells was derived from the results of TLC analysis, showing that prion protein was associated in PrPC immunoprecipitates with GM3, which is the main ganglioside constituent of microdomains in these cells. These results demonstrate a direct physical association of PrPC with ganglioside GM3 within microdomains of lymphocytic cells, suggesting that PrPC represents a structural component of the multimolecular signaling complex involved in T cell activation and other dynamic lymphocytic plasma membrane functions.

Phospholipase D1 is phosphorylated and activated in human airway epithelial cells stimulated by sphingosine-1-phosphate: role of src tyrosine kinase and protein kinase C-D.

Anna Ghelli, Anna Maria Porcelli, Annalisa Facchini*, Silvana Hrelia*, Flavio Flamigni* and Michela Rugolo
Dipart. di Biologia Ev.Sp. and *Dipart. di Biochimica “G.Moruzzi”, Università di Bologna

Sphingosine-1-phosphate (SPP) belongs to a group of lipid mediators that regulate cell proliferation, differentiation and survival [Hla, T. et al. 1999, Biochem. Pharmacol. 58, 201-207]. SPP exhibits high affinity with the edg subfamily of G protein-coupled receptors [Fukushima, N. et al. 2001, Annu. Rev. Pharmacol. Toxicol. 41, 507-534], which comprises edg-1, edg-3, edg-5 (also called AGR16 or H218), edg-6 and edg-8. These receptors are coupled differentially via Gi, Gq and G12/13 and Rho to multiple effector systems including adenylate cyclase, phospholipase C (PLC) and D (PLD), intracellular Ca2+ elevation, extracellular signal regulated kinases, actin stress fibres remodelling, etc.. In a human airway epithelial cell line (CFNPE9o-), we have previously reported that SPP induced elevation of cytosolic Ca2+ and PLD-dependent activation of stress fibres formation. All these effects were sensitive to pertussis toxin treatment, suggesting the involvement of a Gi type of G protein [Orlati,S. et al. 2000, Arch. Biochem. Biophys. 375, 69-77; Porcelli et al., 2002, Cell Signalling 14, 75-81]. Two mammalian PLD enzymes PLD1 and PLD2 have been cloned and shown to have a broad tissue distribution. Both enzymes prefer phosphatidylcholine as substrate and require phosphatidylinositol 4,5-bisphosphate for activity. PLD1 is activated by low molecular weight G proteins either of the Rho family or ADP ribosylation factor (ARF), by protein kinase C (PKC), and can also be tyrosine phosphorylated. In contrast, purified PLD2 is not stimulated by ARF, Rho and PKCa or b, but it can be tyrosine phosphorylated [Liscovitch, M. et al., 2000 Biochem.J. 345, 401-415]. Despite it is known that PKC is involved in the activation of PLD in intact cells, the role of the different PKC isoforms is still poorly understood.
In this study the regulatory role of the PKC-d isoform was revealed by use of the specific inhibitor rottlerin, in combination with antisense oligodeoxynuclotide to PKCd. Cell treatment with antisense oligodeoxynuclotide, but not with sense oligodeoxynuclotide, completely abolished PKCd expression and caused strong inhibition of SPP-stimulated phosphatidic acid formation. Indeed, among the PKC a, b, d, e and x isoforms expressed in these cells, only PKCd was activated upon cell stimulation with SPP, as indicated by translocation into the Triton X-100-soluble membrane fraction. Furthermore, pertussis toxin and genistein abolished both PKCd translocation and PLD activation. In particular, a significant reduction of phosphatidylbutanol formation by SPP was observed in the presence of PP1, an inhibitor of Src tyrosine kinase, and, furthermore, the activity of Src kinase was increased by SPP and inhibited by PP1. However, the level of PKCd tyrosine phosphorylation was not increased in SPP-stimulated cells, suggesting that Src did not directly phosphorylate PKCd. Finally, the level of serine phosphorylation of PLD1 and PLD2 isoforms was not changed, whereas threonine phosphorylation of the PLD1 isoform only was significantly increased in SPP-treated cells, but not in the presence of rottlerin. In conclusion, in CFNPE9o- cells SPP interacts with an edg-family membrane receptor linked to a Gi type of G protein, leading to activation of the PLD1 isoform, through a signalling pathway involving Src and PKCd.

Inhibition of human PBL proliferation by inhibitors of the sphingolipid pathway.

Lucia Cavallini and Adolfo Alexandre
Dipartimento di Chimica Biologica, Università di Padova

The activation of T lymphocytes is the central event in the development of immunity. IL-2 is produced early upon stimulation, together with the high affinity IL-2 receptor, (CD25). They mediate proliferation and differentiation responses, as well as apoptosis at the end of the process of clonal expansion.
Two independent studies report the involvement of an acidic sphingomyelinase (SMase) in ceramide production in the CD28 costimulary signalling of T cell (Boucher (1995) J Exp Med , 181, 2059), and of a neutral fumonisin B1-sensitive SMase in a T cell hybridoma (Tonnetti (1999) J. Exp. Med. 189, 1581). Since in many instances the destiny of ceramide is to be deacylated to sphingosine and then phosphorylated to sphingosine-1P (SPP) we studied the role of these sphingolipid derivatives on the lymphocyte responses to activating stimuli. We performed experiments to test the effect of FumB1, a sphingosine syntase inhibitor but also neutral sphingomyelinase inhibitor, D-erythro-MAPP an inhibitor of neutral ceramidase and N,N-dimethylsphingosine (DMS), a competitive inhibitor of sphingosine kinase(SPHK) on lymphocyte proliferation induced by phytohemoagglutinin (PHA) and found a strong inhibition by these agents. These findings suggest the involvement of SPP in lymphocyte activation. Inhibition is not relieved by addition of exogenous C2-ceramide, sphingosine or SPP in a large concentration range. All reagents induce a paradoxical increase of inhibition. Sphingosine or ceramide however could act as pro-apoptotic agents and SPP could not enter the cell or act through specific inhibitory receptors (Edg family).
SPP seems slightly increased in PBL after 24 hours of stimulation. To address the involvement of SPP in T cell activation we tried to inhibit SPHK expression by means of five different antisense oligonucleotides directed toward human SPHK isoform-1. An inhibitory effect of 10% was effectively obtained at 10 uM concentration with two of the 24mers tested but the data are not significant due to the interferences of the oligonucleotide content of thimidine in the 3H thimidine incorporation. Other methods less sensitive and reliable as the MTT or Alamar-blue tests tend to confirm this data. We also measured the activation of the SPHK in the extract of PHA stimulated PBL after various times and found a net increase of the SPHK activity at 24 hours. This information together with the increase of SPP and with the inhibition by DMS prove the involvement of SPP in the mitogenic response of T cell.
The late increase of SPP and SPHK activity in PHA stimulated PBL indicate their involvement also in cell cycle control following TCR stimulation. Indeed adding DMS 1 hour after stimulation does not abolish the inhibition of proliferation. Furthermore DMS has no appreciable effect on NFkB activation, and also the production of IL-2 and release of soluble CD25 are only marginally inhibited by DMS (decreases ranging from 20-40% compared to the complete inhibition of the proliferation).
The DMS inhibition (75-100%) of proliferation is also observed in IL-2-dependent proliferation of PBL pre-stimulated with PHA for 74 hours, then washed and rested in low serum. The inhibition is accompanied by inhibition of pRb phosphorylation and cyclin A increase .

Free and cell-associated ganglioside interaction differently affects the biological activity of fibroblast growth factor-2 (FGF-2).

Marco Rusnati, Chiara Urbinati and Marco Presta
Sezione di Patologia Generale, Dipartimento di Scienze Biomediche e Biotecnologie, Facoltà di Medicina, Università di Brescia

Gangliosides, normally associated to cell membranes, are shed in extracellular environment during tumor growth. Also, gangliosides regulate tumor neovascularization that occurs through the stimulation of endothelial cells by angiogenic growth factors such as fibroblast growth factor-2 (FGF-2). In vitro, FGF-2 induces proliferation of endothelial cells by interacting with high affinity tyrosine kinase receptors (FGFRs) and low affinity heparan-sulfate proteoglycans (HSPGs) of the cell surface. Here we report about the ability of both free and cell-associated gangliosides to bind FGF-2 and to modulate its cell-interaction and biological activity.
Free gangliosides: Size exclusion chromatography demonstrates that native, but not heat-denatured FGF-2 binds to GT1b, GD1b or GM1 ganglioside. Also, gangliosides protect native FGF-2 from trypsin digestion (the order of relative potency being: GT1b>GD1b>GM1=GM2>GM3= galactosyl-ceramide) whereas asialo-GM1 (aGM1), free NeuAc, and N-acetylneuramin-lactose are ineffective. Thus, FGF-2 gangliosides interaction occurs through NeuAc residue(s) but it is modulated by the oligosaccharide chain and the ceramidic portion of the glycosphingolipid. Scatchard plot analysis of the binding data of fluorochrome-labeled GM1 (BodyPy-GM1) to immobilized FGF-2 indicates that FGF-2/GM1 interaction occurs with a Kd equal to 1-6 ?M. By evaluating the capacity of synthetic FGF-2 peptide fragments to prevent the binding of BodyPy-GM1 to immobilized FGF-2, the COOH-terminus of FGF-2 has been identified as the putative ganglioside-binding region. Gangliosides inhibit the binding of FGF-2 to FGFRs but not to HSPGs of endothelial GM 7373 cells (the order of relative potency being GT1b>GD1b>GM1>aGM1). Accordingly, gangliosides inhibit the mitogenic activity exerted by FGF-2 in GM 7373 cells. Also in this case, GT1b is the most effective among the gangliosides tested while aGM1, free NeuAc, N-acetylneuramin-lactose, and galactosyl-ceramide are ineffective.
Cell associated gangliosides: Treatment of GM 7373 cells with ganglioside biosynthesis inhibitors fumonisin B1, PDMP or PPPP impairs their capacity to proliferate when exposed to FGF2. Also, the mitogenic activity of FGF2 is inhibited by the GM1-binding cholera toxin B subunit (CTB). Conversely, overloading of GM 7373 cell membranes with exogenous GM1 causes a 10-fold increase of the mitogenic potency of FGF2. 125I-FGF2 binds to cell membrane GM1 (Kd = 3 nM) in complex ganglioside/heparan sulfate-deficient CHO-K1-pgsA745 cell mutants that were overloaded with exogenous GM1. Moreover, FGF2 competes with FITC-CTB for the binding to cell membrane GM1 in different CHO cell lines independently of their capacity to express heparan sulfate proteoglycans. Conversely, CTB inhibits cell proliferation triggered by FGF2 in CHO cells overexpressing the tyrosine kinase FGF receptor-1 (FGFR1). Finally, GM1-overloading confers to FGFR1-transfected, complex ganglioside-deficient CHO-K1 cell mutants the capacity to proliferate when stimulated by FGF2. This is inhibited by CTB. Cell proliferation triggered by serum or by TPA is instead independent of the cell membrane ganglioside milieu.
Here we demonstrate that FGF-2 binds both free and cell-associated gangliosides. The distinct interaction leads however to opposite effects. Indeed, free gangliosides act as FGF-2 antagonists by sequestering the growth factor in the extracellular environment and preventing cell interaction. On the contrary, cell-associated gangliosides act as co-receptors for FGF-2 and their interaction with the growth factor is required for cell proliferation.

Nitric oxide in glioma cell growth: role of ceramide.

P. Viani, P. Giussani, L. Brioschi, L.Riboni, G. Tettamanti
Department of Medical Chemistry and Biochemistry, University of Milan

Many literature reports indicate ceramide and nitric oxide (NO) as crucial modulators of the cell fate in the nervous system. In fact in cells from the nervous system ceramide and NO are both able to regulate important biological events such as cell growth, differentiation and apoptosis possibly acting as modulators of the related signal transduction pathways. Notwithstanding these functional relationships very little is known on the possible role of ceramide in the NO-mediated signaling in the nervous system. In our laboratories we obtained evidence that NO acts as an antiproliferativee agent in C6 glioma cells. This is associated to an increase of cellular ceramide and can be mimicked by treatments that augment intracellular levels of ceramide. Different metabolic studies indicate the NO-mediated ceramide accumulation is due to its reduced utilization for the biosynthesis of complex sphingolipids. Notwithstanding this evidence the enzymatic activity of both sphingomyelin synthase and glucosylceramide synthase are not modified by NO. On these basis we investigated the possibility that the availability of ceramide for the biosynthesis of complex sphingolipids is impaired by NO. The effect of NO on the metabolic utilization of N-hexanoyl- [3H]sphingosine by C6 glioma cells demonstrate that the biosynthesis of N-hexanoyl-complex sphigolipids which does not require ceramide trafficking from endoplasmic reticulum to Golgi apparatus, is not affected. On the contrary the utilization of [3H]ceramide derived from recycled [3H]sphingosine, produced from N-hexanoyl- [3H]sphingosine catabolism, is strongly inhibited by NO. Different experimental approaches that influence the traffic between endoplasmic reticulum and Golgi apparatus indicate that the transport possibly vesicle-mediated of ceramide between these subcellular compartments is impaired by NO. As a consequence newly synthesized ceramide accumulates and appears to act as a mediator of the antiproliferative effect of NO. In conclusion these results demonstrate that in glioma cells, the modulation of ceramide intracellular traffic can contribute to the regulation of the intracellular levels of this sphingoid mediator and participate to ceramide-mediated signaling pathways.

DNA damage in human fibroblasts exposed to fumonisin B1.

F. Galvano(1), L. La Fauci(1), A. Russo(2), A. Campisi2, A. Vanella(2) and M. Renis(2)
(1) Department of Agro-forestry, Environmental Science and Technology, University of Reggio Calabria; (2) Department of Biochemistry, Medical Chemistry and Molecular Biology, University of Catania

Fumonisins are mycotoxins produced by several Fusarium species (Fusarium verticilloides and Fusarium proliferatum) that infest corn and other cereals. Among several classes of fumonisins (A, B, or C) fumonisin B1 (FB1) is the most prevalent and has higher toxicological significance because it is frequently present at high concentrations on both diseased and apparently healthy corn plants. FB1 ingestion as the purified compound as well as F. moniliforme-contaminated corn is linked to a wide spectrum of severe animal diseases and patho-physiologic effects such as equine neurodegenerative disease named “leukoencephalomalacia”, porcine pulmonary oedema, immunosuppression in poultry and nephrotoxicity in sheep, toxicity in fish, atherosclerosis and cardiac thrombosis in non-human primates, cardiovascular toxicity in swine and liver toxicity and carcinogenicity in rats and mice. In addition, epidemiological studies in some areas of South Africa and China evidenced a correlation between the ingestion of FB1 contaminated food and higher incidence of oesophageal carcinoma in humans. FB1 structurally resembles sphingoid bases and acts as inhibitor of ceramide synthetase, a key enzyme involved in de novo sphingolipid biosynthesis and in the reacylation of free sphingoid bases derived from sphingolipid turnover. This inhibitory effect leads to accumulation of free sphinganine and sphingosine and subsequent induction of cell death. However, the down stream effectors activated by these sphingolipids in the cell death-signalling pathway are little known. The aim of this study was to evaluate, in FB1 exposed human fibroblasts, the involvement of oxygen free radicals and of some other biochemical pathways, caspase-3 activity, poly(ADP-ribose)polymerase (PARP) cleavage and DNA damage evaluated by COMET assay. Our results indicate that FB1 treatment (48, 72 h and 10, 50, 100 mM) does not affect cellular viability. Conversely, after 72 h of treatment, FB1 (50 and 100 mM) induced DNA damage, an enhancement of caspase-3-activity and cleavage of poly(ADP-ribose)polymerase compared to controls. In addition, FB1 increased the expression of HSP70 in a concentration and time-dependent manner. Our results indicate that DNA damage of apoptotic type in human fibroblasts is caused by exposure to FB1 at high concentrations and for prolonged time and that the genotoxic potential of FB1 has probably been underestimated and should be reconsidered.

Apoptosis in HT-29 colon cancer cells by ceramide: temporal relationship with impairment of the transport and processing of pro-cathepsin D.

Daniela De Stefanis(1), Patrizia Reffo1, Gabriella Bonelli(1), Francesco M. Baccino(1), Giusy Sala(2), Riccardo Ghidoni(2), Patrice Codogno(3) and Ciro Isidoro(4)
(1) Dipartimento di Medicina e Oncologia Sperimentale, Università di Torino
(2) Laboratorio di Biochimica, Ospedale San Paolo, Università degli Studi di Milano
(3) INSERM U504, Villejuif, France
(4) Università “A. Avogadro”, Dipartimento di Scienze Mediche, Novara

Ceramide has been suggested as an important mediator of apoptosis. The effect of increased intracellular levels of ceramide on the transport and processing of cathepsin D (CD), a lysosomal protease implicated in apoptosis of tumour cells, was investigated in HT-29 human colorectal cancer cells. Lysosomal targeting of proCD was inhibited in a dose- and time-dependent manner by exogenous N-Acetylsphingosine (NAS, also known as C2-ceramide), but not by its C2-dihydroceramide (DH-C2) inactive analogue. This effect of NAS was mimicked, at least in part, by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol (PDMP), which inhibits the metabolic conversion of endogenous ceramide into sphingolipids. Both these drugs effectively induced an increase of intracellular ceramide levels of 3.5-folds and of about 8-folds over basal levels within 8 h and 24 h of incubation, respectively. The treatment with either NAS or PDMP revealed to be cytotoxic for HT-29 cells and lead to cell death with classical characteristics of apoptosis including cytosolic release of cytochrome c from mithocondria and cell surface exposition of annexin V binding sites. Morphological signs of apoptosis and DNA fragmentation became apparent, however, only between 24 and 48 h of incubation and poly(ADP ribose)-polymerase cleavage, a hallmark of caspase 3 activity, occurred not earlier than 8 h from incubation. Caspase 3 inhibition protected, whereas CD inhibition by pepstatin A did not, from NAS-induced apoptosis, indicating that once high levels of ceramide have accumulated in the cell CD-mediated proteolysis is not required for subsequent reactions leading to apoptosis. The above-described effects of NAS and PDMP on the transport and maturation of proCD were apparent already 2 h after incubation with the drugs, which is much earlier than when classical biochemical and morphological evidences of apoptosis could be detected. This suggests that impairment of glycoproteins transport along the secretory pathway due to increased levels of cell-associated ceramide is an early event in cells undergoing apoptosis. Of note, secretion of proCD was almost abolished and formation of double-chain mature CD was reduced and delayed by NAS, whereas PDMP did not affect much proCD secretion, though it largely inhibited the endosomal-lysosomal segregation of this molecule.

Serum deprivation increases ceramide levels and induces apoptosis in embryonic hippocampal HN9.10e cells.

Laura Colombaioni(1), Luisella Colombini(2), Laura M. Frago(3), Isabel Varela-Nieto(3), Rossana Pesi(2) and Mercedes Garcia-Gil(2)
(1) Institute of Neurophysiology of CNR, Pisa
(2) Dpt. of Physiology and Biochemistry, University of Pisa
(3) Instituto de Investigaciones Biomedicas Alberto Sols, CSIC-UAM, Madrid 28029, Spain

Sphingolipid metabolites have been involved in the regulation of proliferation, differentiation and apoptosis. While cellular mechanisms of these processes have been extensively analysed in postmitotic neurones, little is known about proliferating neuronal precursors. We have taken as a model of neuroblasts the embryonic hippocampal cell line HN9.10e. In undifferentiated cells, apoptosis was induced by serum deprivation and by treatment
with N-acetylsphingosine (C2-Cer). The intracellular levels of ceramide peaked at one hour following serum deprivation. We observed translocation of Bax from cytosol to mitochondria after one hour of serum withdrawal followed, two hours later, by cytochrome c release from mitochondria. These events occurred without mitochondrial membrane potential loss nor mitochondrial calcium raise. As calcium is implicated in several cell death pathways, we analysed intracellular calcium levels after longer periods of serum deprivation. After six hours, an increase of cytosolic Ca++ was detected in HN9.10e cells loaded with the Ca++ indicator Fluo3-AM. We have also measured caspase-3 activity and we have found caspase-3 activation after 24 hours but not after 4 hours of serum deprivation or C2-Cer treatment. Cells serum-deprived for four hours and then grown in complete medium for twenty hours fully recovered viability. Summarising, in HN9.10e cells, apoptosis involves increases in ceramide levels, translocation of Bax, release of cytochrome c, and maintenance of mitochondrial functionality followed by calcium deregulation. The lack of caspase-3 activation, in addition to the maintenance of mitochondrial function could allow the reversibility of death commitment and provide neuroblasts a longer temporal window to decide their fate.

The proapoptotic effect of retinoic acid in breast cancer cells requires RARb and is mediated by increased endogenous ceramide levels.

Giulia Somenzi(1), Silvia Pozzi(1), Giusy Sala(2), Riccardo Ghidoni(2), Nicoletta Sacchi(1)
Labs of Molecular Genetics(1) and Biochemistry(2),
San Paolo University Hospital, School of Medicine, University of Milan

Retinoids are important regulators of cell proliferation, differentiation and apoptosis. One of the most active retinoids is all-trans-retinoic acid (ATRA) that is being evaluated as chemopreventive and chemotherapeutic agent for many cancers, including breast cancer. It is well known that the molecular action of ATRA is mediated by nuclear receptors (RARs a, b, g and RXRs a, b, g), and that RARb silencing is one of the main reasons for breast cancer resistance towards treatment with retinoids. The ATRA mechanism of action as a proapoptotic agent is largely unknown.
Here we focus on the possibility that endogenous ceramide is one of the molecular targets of ATRA in breast cancer cells, as it was suggested for other cell lines (neuroblastoma and prostate carcinoma cells) and that the mechanism of action is influenced by the nuclear receptor status of the cell line. For this purpose we used two breast cancer cell lines, T47D cells that have no basal expression of RAR?, but that can re-express this receptor after treatment with pharmacological doses of ATRA, and MDA-MB-231 cells, that have a lack of transcription of RAR?, due to high methylation of the CpG islands in its promoter. In addition, T47D cells express high levels of RAR?, that is known to promote RAR? transcription, whereas MDA-MB-231 cells do not.
We found that ATRA (1 ?M, 48 h) induced two-fold endogenous ceramide accumulation in T47D cells, whereas no ceramide increase was observed in MDA-MB-231, after the same treatment. Endogenous ceramide increase correlated with the antiproliferative effect of the drug: ATRA treatment concomitantly impaired cell growth, colony formation and promoted apoptosis. Conversely, no biological effect was observed in MDA-MB-231 cells. Concomitant treatment of ATRA and RARs antagonists resulted in lack of ceramide accumulation and biological effect, thus suggesting that the ATRA signaling pathway requires RAR? expression and is mediated by endogenous ceramide accumulation.

Sialic acid acetylation suppresses the pro-apoptotic activity of GD3.

F. Malisan*, B. Tomassini*, L. Franchi*, N. Ventura*, M.R. Rippo*, I. Condo’*, L. Liberati*, A. Rufini*, C. Nachtigall^, B. Kniep^ and R. Testi*.
*Laboratory of Immunology and Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Roma
^ Institute of Immunology, Technical University of Dresden, Germany.

GD3 disialoganglioside acts as an intracellular mediator of apoptosis being responsible for the opening of the mitochondrial permeability transition pore, the release of cytochrome c and the activation of caspase-9. Sialic acid 9-O-acetylation, the most common post-synthetic modification of GD3, affects the physicochemical properties of the parent molecule and is expected to modify its functions. We investigated whether sialic acid 9-O-acetylation affects the pro-apoptotic function of GD3.
Mitochondria are a primary target for GD3. Interestingly, 9-O-acetyl-GD3, unlike GD3, is completely unable to induce cytochrome c release and caspase-9 activation from isolated mitochondria. In addition, whereas exogenous GD3 directly triggers apoptosis of HEK293 cells, exogenous 9-O-acetyl-GD3 can not. Moreover, while most cell types undergo apoptosis upon GD3 synthase overexpression due to endogenous GD3 synthesis and accumulation, the cell line HEK293 is resistant to GD3 synthase-mediated apoptosis. We observed that these cells, upon GD3 synthase overexpression, converted part of the GD3 into 9-O- acetyl-GD3. Finally, when HEK293 cells are co-transfected with GD3 synthase and a 9-O-acetyl-esterase, to prevent 9-O-acetyl-GD3 accumulation, they undergo massive apoptosis.
These data indicate that sialic acid 9-O-acetylation suppresses the pro-apoptotic activity of GD3 and suggest a new role for sialic acid acetylation in the control of the apoptotic program.

Selective induction of cell death by disialogangliosides through opening of the mitochondrial permeability transition pore.

Luca Scorrano(a), Maria Eugenia Soriano(a), Gunter Kirschner(b), Paolo Bernardi (a)
(a) Department of Biomedical Sciences, University of Padova
(b) Fidia Research Laboratories, Abano Terme

Mitochondria amplify cell death signals releasing protein cofactors essential for the activation of effector caspases. Certain lipid second messengers including arachidonic acid and ganglioside GD3 trigger this release by inducing the mitochondrial permeability transition (PT). To gain further insight into the specificity of mitochondrial effects of gangliosides, we compared a and b-series gangliosides, which differ for the number of sialic acid.
The b-series disialogangliosides GD3, GD1b, GT1b and GQ1b induced the PT in isolated mitochondria, while a-series monosialogangliosides GM3, GM2, GM1 and GD1a display no effect. GQ1b and GT1b resulted more potent than GD3 and GD1b. Exogenous GT1b caused PT in situ, accompanied by mitochondrial depolarization and followed by CsA-sensitive cell death. Thus, all b-series gangliosides can act as proapoptotic second messengers that induce apoptosis through the mitochondrial pathway. Our data show that two adjacent sialic acid residues are required for gangliosides to induce the PT. This structural specificity may represent a useful template to tailor drugs aimed at induce cell death.

Effects of ganglioside biosynthesis inhibition on the expression and phosphorylation of membrane tyrosine-kinase receptor p185neu/erbb2 in tumor and normal mammary cell lines.

E. Sottocornola, B. Berra and I. Colombo
Institute of General Physiology and Biological Chemistry, University of Milan

The functional relationship between gangliosides and growth factor receptors (GFRs) with tyrosine-kinase activity has already been extensively analysed. So far, no study has been performed in order to define the relation between gangliosides and the oncoprotein p185neu or its normal counterpart erbB2, although their well known involvement in the mammary adenocarcinoma development and in the mammary gland differentiation.
The mouse mammary adenocarcinoma cell line MG1361, derived from mammary tumors of MMTV-neu transgenic mice, and the normal mouse mammary epithelial cell line HC11, in which erbB2 expression is inducible by the cell confluence degree in culture, are the experimental models analysed in the present study.
In the MG1361 cells, p185neu is constitutively in the phosphorylated active form, because of a single aminoacid substitution in the transmembrane domain (Val664Glu). 10 nM EGF was used to activate (auto-phosphorylation) the erbB2 receptor in HC11 cells. Inhibition of ganglioside biosynthesis in both the cell lines was achieved by 30 mM D-PDMP treatment for five days. Gangliosides were analysed by HP-TLC. p185neu and erbB2 expression and phosphorylation were evaluated by western blot.
Our results demonstrate that, in HC11 cells, erbB2 and ganglioside GM3 expression raise together when increasing the degree of confluence of the cells. In EGF-stimulated HC11 cells, erbB2 is auto-phosphorylated, but its expression level decreases, as well as the ganglioside GM3 content, with respect to not-stimulated cells. Expression of other ganglioside species remains unchanged. Inhibition of ganglioside biosynthesis by D-PDMP treatment in EGF-stimulated HC11 cells does not interfere with the erbB2 auto-phosphorylation ability, but it maintains high expression levels of the receptor. On the contrary, in MG1361 cells, inhibition of ganglioside biosynthesis modifies nor p185neu expression neither its phosphorylation levels.
These data give support to the hypothesis of a tight functional relationship between erbB2 and GM3 in HC11 cells. Both the molecules could co-localise at the plasma membrane level and the presence of GM3 could be of importance in the normal internalisation and turn-over of the activated receptor rather than in the receptor activation. It is to be further investigated if these two molecules are located within the glycosphingolipid enriched microdomains (GEM) and, if it is so, the nature of their molecular relationship.
The absence of a correlation between p185neu and ganglioside expression, in MG1361 cells, might be attributed to the nature of the oncoprotein, that is over-expressed and constitutively activated.

β1,3 galactosyltransferase T5: regulation and involvement in the expression of CA19.9 tumor antigen.

Marco Trinchera
Dipartimento di Scienze Biomediche Sperimentali e Cliniche,Università dell’Insubria, Varese

The CA19.9 antigen is a sialyl-Lewis a tetrasaccharide carried by mucins and glycosphingolipids detected in cancer tissues and cell lines. The b 1,3 galactosyltransferase T5 (b 3Gal-T5), an enzyme preferentially acting on glycosphingolipid substrates in vitro, is studied at the molecular level in this research as a candidate key enzyme in CA19.9 biosynthesis. b 3Gal-T5 transcript was first reported to be well detectable only in adenocarcinoma cell lines expressing CA19.9, but not expressed or present at very low levels in the lines lacking the antigen. More recently, we found that the transcript is strongly down-regulated (over 30-folds) in colon adenocarcinomas versus the normal colon mucosa, and other authors reported that even the enzyme activity is down-regulated in colon adenocarcinomas, but less strongly (about 5-folds). These data are paradoxical since CA19.9 biosynthesis is enhanced in cancer with respect to the normal tissue, and in fact it is widely used as a marker in the diagnosis and follow-up of adenocarcinomas of the gastrointestinal tract. Two possible alternative hypotheses to explain the data are: 1) the presence of a novel b 3Gal-T isoform, unknown at the present, differentially expressed in native cancers, and 2) the possibility that b 3Gal-T5 is not primarily regulated at the transcriptional level. To address this issue, we looked for cellular models resembling native adenocarcinomas and found that the gastric adenocarcinoma cell line MKN-45 expresses low levels of b 3Gal-T5 transcript and no CA19.9 antigen, but strong b 3Gal-T activity. In addition, recombinant MKN-45 clones obtained by Fuc-TIII transfection express very high levels of CA19.9 too. Moreover, in the human pancreas adenocarcinoma cell line BxPc3, CA 19.9 antigen and b 3Gal-T5 activity are both well detected, while b3Gal-T5 transcript is really faint, and in other cell lines where b 3Gal-T5 transcript is more abundant than in BxPc3 cells, the b 3Gal-T5 activity is not higher than in BxPc3. In particular, in recombinant HCT-15 cells expressing high levels of b 3Gal-T5 transcript, due to the strong CMV promoter, b 3Gal-T5 activity is comparable to that measured in BxPc3 cells, and thus much lower than in MKN-45. The above results suggest that transcript levels alone are not predictive of b 3Gal-T activity. To rule out the possibility of multiple b 3Gal-T isoforms, we planned to determine the enzyme kinetics in native or recombinant cell lines as well as in normal colon mucosa or adenocarcinomas. Results available by now show that the Km values calculated with MKN-45 or recombinant cells expressing true b 3Gal-T5 are identical toward three different substrates, as well as toward donor UDP-Gal, and undistinguishable from those reported in human normal mucosa or adenocarcinomas, suggesting that another b3Gal-T isoform is not probably involved. To prove that b 3Gal-T5 alone is responsible for b 3Gal-T activity and CA19.9 biosynthesis in cells where the transcript level is low, we are now trying to suppress it in recombinant MKN-45/Fuc-TIII and BxPc3 clones by expression of an antisense b 3Gal-T5 fragment.

α-galactoside α2,6 sialyltransferase, the enzyme responsible for the α2,6 sialylation of N-acetyllactosaminic chains in glycoproteins and glycolipids, is up-regulated in human cancers.

Fabio Dall’Olio and Mariella Chiricolo
Dipartimento di Patologia Sperimentale, Università di Bologna

α-galactoside α2,6sialyltransferase (ST6Gal I) is the only enzyme which can mediate the transfer of sialic acid in a2,6 linkage to N-acetyllactosamine (Gala1,4GlcNAc), a structure commonly found in glycoproteins and glycosphingolipids [1]. The main regulatory mechanisms of ST6Gal.I expression operate transcriptionally, through the use of distinct promoters which results in the expression of mRNA species differing in the 5′ untranslated regions (reviewed in [2]). In several malignancies, including colon and breast cancer, ST6Gal.I shows a dramatic up-regulation [3]. In colon cancer, where this phenomenon is in part mediated by a transcriptional elevation through the promoter which is usually utilized by liver cells [4,5] , the extent of the upregulation of ST6Gal.I and of the α2,6-linked sialic acid on the cell surface is an indicator of a poor prognosis [6,7]. The relationship between ST6Gal I elevation and neoplastic transformation has been investigated under two aspects. First, the phenotypic changes induced by ST6Gal.I overexpression have been studied upon transfection of the ST6Gal I cDNA in colon cancer cell lines devoid of such enzyme activity [8,9]. The phenotypic changes observed are mild and appear to be dependent on the cell type. Second, the effect of ras and neu oncogenes overexpression on ST6Gal I modulation has been investigated in murine fibroblasts and breast cancer cells respectively. Transfection of both K-ras and H-ras in NIH3T3 fibroblasts results in a dramatic transcriptional activation of the gene and a consequent appearance of α2,6-sialylated glycans at the cell surface. Murine breast cancer cell lines have been obtained from spontaneously developed breast tumors in animals constitutivelly expressing the neu oncogene. Among the cell lines obtained, those expressing neu showed a strong ST6Gal I expression, while revertant cell lines not expressing neu showed negligible ST6Gal I expression. These data indicate that the transcription of ST6Gal I gene is controlled by protein kinase pathways and provide a possible link between ST6Gal I upregulation and invasivity of human cancers.
References
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4. Dall’Olio F, Chiricolo M, Lau JT, Int J Cancer 81, 243-7 (1999).
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6. Lise M, Belluco C, Perera SP, Patel R, Thomas P, Ganguly A, Hybridoma 19, 281-6 (2000).
7. Vierbuchen MJ, Fruechtnicht W, Brackrock S, Krause KT, Zienkiewicz TJ, Cancer 76, 727-35 (1995).
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Involvement of rat cytosolic sialidase neu2 in differentiation and proliferation of PC12 cells.

A. Fanzani, R. Giuliani, D. Zizioli and S. Marchesini
Sezione di Bicohimica, Dipartimento di Scienze Biomediche e Biotecnologie, Facoltà di Medicina, Università di Brescia

Cytosolic sialidase Neu2 is a protein of 379 amminoacids that is highly expressed in rat skeletal muscle and that contains an enhancer/promoter region which is transcriptionally active in rat L6 myogenic cells.
An increase in cytosolic sialidase transcript has been observed by means of RT-PCR during differentiation of PC12 cells, as well as during maturation of rat cerebellar granule cells, two models of neuronal differentiation. In particular, PC12 cells treated with differentiating agents such as NGF and bFGF, showed an increase of Neu2 mRNA up to 3 and 2 days, respectively. Interestingly, we observed also an increment of the transcript with EGF, a growth factor that induces proliferation. To obtain further informations on the sialidase expression we cloned a fragment of 1.4 kb which is sufficient in rat L6 mioblasts to activate the transcription in transient transfections.
Experiments of transient transfections in PC12 cells have confirmed a trascriptionally activation of the promoter with NGF and bFGF. The sequence analysis of the promoter has shown that the presence of two E-box motifs could be important for the activation, together with others tipical sites involved in NGF activation. The cDNA of Neu2 has been cloned in a inducible vector for the stable transfection of PC12 cells, and we are working to produce a stable cell line for the antisense oligo expression. From these experiments we hope to have informations on the Neu2 involvement in the differentiation process. In order to purify enough Neu2 protein for in vitro enzimatic studies, a fusion protein GST-Neu2 is under preparation.

Ceramide levels are inversely associated with malignant progression of human glial tumors.

Riboni L.(1), Campanella R.(2), Bassi R.(1), Viani P.(1), Tettamanti G.(1)
(1) Dpt. of Medical Chemistry and Biochemistry, University of Milan, LITA-Segrate, Milano
(2)  Neurosurgical Operative Unit, Dpt. of Neurological Sciences, University of Milan, IRCCS Ospedale Maggiore Policlinico, Milano

Astrocytomas represent the most frequent and deadly primary intracranial neoplasias in humans. On the basis of their histopathological and clinical features, these tumors can occur in different stages of malignancy, ranging from low- to high-grade tumors. Although some late stage tumors seem to arise de novo, follow-up studies of patients with low or medium stage disease have shown that most will progress over time to a higher stage, supporting malignant progression in vivo. To determine whether ceramide levels correlate with the malignant progression of human astrocytomas, we investigated these levels in surgical specimens of glial tumors of low- and high-grade malignancy. Tumor samples, obtained from 52 patients who underwent therapeutic removal of primary brain tumors were used. The tumors were classified according to standard morphologic criteria and they were grouped into tumors with low-grade and high-grade of malignancy. Sections of normal brain tissue adjacent to the tumor were also analyzed in 11 of the 52 patients. Ceramide levels were significantly lower in the combined high-grade tumors compared to low-grade ones and in both tumor groups compared to peritumoral tissue. The results obtained indicate an inverse correlation between the amount of ceramide and tumor malignancy evaluated by both histological and biochemical parameters. Moreover, the highest ceramide levels were found in patients with a longer survival time. Altogether, the present findings indicate that ceramide is inversely associated with malignant progression of human astrocytomas and poor prognosis. The down regulation of ceramide levels in human astrocytomas emerges as a novel alteration that may contribute to glial neoplastic transformation. The low ceramide levels in high-grade tumors may underlie their rapid growth and apoptotic resistant features. This study supports the rationale for the potential benefits of a ceramide-based chemotherapy.