A role for the de novo sphingolipids in apoptosis of photosensitized cells

Sphingolipids have been implicated in apoptosis after various stress inducers. To assess the involvement of the de novo sphingolipid pathway in apoptosis, photodynamic therapy (PDT) with the photosensitizer Pc 4 was used as a novel stress inducer. Here we provide biochemical and genetic evidence of the role of the de novo sphingolipids in apoptosis post-Pc 4-PDT. In Jurkat cells PDT-induced intracellular sphinganine accumulation, DEVDase activation, PARP cleavage, and apoptosis were suppressed by the de novo sphingolipid synthesis inhibitor ISP-1 (Myriocin). Coincubation with sphinganine, sphingosine, or C16-ceramide specifically reversed the antiapoptotic actions of ISP-1 or the singlet oxygen scavenger L-histidine. PDT-induced cytochrome c release from mitochondria into the cytosol was inhibited by L-histidine, but not by ISP-1. Cotreatment with sphinganine did not reverse the inhibitory effect of L-histidine. In addition, PDT-induced sphinganine accumulation and apoptosis were ISP-1-sensitive in A431 human epidermoid and HT29 human carcinoma cells. Furthermore, in LY-B cells, CHO-derived mutants deficient in the de novo sphingolipid synthesis enzyme serine palmitoyltransferase (SPT) activity, DEVDase activation and apoptosis were delayed and suppressed post-PDT. Hence, the data are consistent with the partial involvement of the de novo sphingolipid pathway in apoptosis via DEVDase activation downstream of mitochondrial cytochrome c release post-Pc 4-PDT.     

De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1.

Previous studies have demonstrated that several splice variants are derived from both the caspase 9 and Bcl-x genes in which the Bcl-x splice variant, Bcl-x(L) and the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9. In a recent study, we showed that ceramide induces the dephosphorylation of SR proteins, a family of protein factors that regulate alternative splicing. In this study, the regulation of the alternative processing of pre-mRNA of both caspase 9 and Bcl-x(L) was examined in response to ceramide. Treatment of A549 lung adenocarcinoma cells with cell-permeable ceramide, D-e-C(6) ceramide, down-regulated the levels of Bcl-x(L) and caspase 9b mRNA and immunoreactive protein with a concomitant increase in the mRNA and immunoreactive protein levels of Bcl-x(s) and caspase 9 in a dose- and time-dependent manner. Pretreatment with calyculin A (5 nm), an inhibitor of protein phosphatase-1 (PP1) and protein phosphatase 2A (PP2A) blocked ceramide-induced alternative splicing in contrast to okadaic acid (10 nm), a specific inhibitor of PP2A at this concentrations in cells, demonstrating a PP1-mediated mechanism. A role for endogenous ceramide in regulating the alternative splicing of caspase 9 and Bcl-x was demonstrated using the chemotherapeutic agent, gemcitabine. Treatment of A549 cells with gemcitabine (1 microm) increased ceramide levels 3-fold via the de novo sphingolipid pathway as determined by pulse labeling experiments and inhibition studies with myriocin (50 nm), a specific inhibitor of serine palmitoyltransferase (the first step in de novo synthesis of ceramide). Treatment of A549 cells with gemcitabine down-regulated the levels of Bcl-x(L) and caspase 9b mRNA with a concomitant increase in the mRNA levels of Bcl-x(s) and caspase 9. Again, inhibitors of ceramide synthesis blocked this effect. We also demonstrate that the change in the alternative splicing of caspase 9 and Bcl-x occurred prior to apoptosis following treatment with gemcitabine. Furthermore, doses of D-e-C(6) ceramide that induce the alternative splicing of both caspase 9 and Bcl-x-sensitized A549 cells to daunorubicin. These data demonstrate a role for protein phosphatases 1 (PP1) and endogenous ceramide generated via the de novo pathway in regulating this mechanism. This is the first report on the dynamic regulation of RNA splicing of members of the Bcl-2 and caspase families in response to regulators of apoptosis.     

Crm-A, bcl-2 and NDGA inhibit CD95L-induced apoptosis of malignant glioma cells at the level of caspase 8 processing

Susceptibility to CD95 (Fas/APO-1)-mediated apoptosis in human glioma cells depends on CD95 expression and unknown factors that regulate signal transduction. Thus, LN-18 cells are highly sensitive to CD95 ligand (CD95L) whereas LN-229 cells require coexposure to inhibitors of RNA or protein synthesis for induction of apoptosis. Here, we report that caspase 8 and 3 activation, poly(ADP-ribose)polymerase cleavage and apoptosis are inhibited by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), or ectopic expression of crm-A or bcl-2. CD95L-induced glioma cell apoptosis does not involve ceramide generation. Apoptosis induced by exogenous ceramide resembles CD95-mediated apoptosis in that bcl-2 is protective but differs in that NDGA and crm-A have no effect and in that cycloheximide (CHX) inhibits rather than potentiates ceramide-induced cell death. We conclude that caspase 8 and caspase 3 activation, but not ceramide generation, are required for CD95 ligand-induced apoptosis of glioma cells and that bcl-2, crm-A and NDGA all act upstream of caspases to inhibit apoptosis.     

Involvement of de novo ceramide synthesis in pro-inflammatory adipokine secretion and adipocyte–macrophage interaction

Interaction between adipocytes and macrophages has been suggested to play a central role in the pathogenesis of obesity. Ceramide, a sphingolipid de novo synthesized from palmitate, is known to stimulate pro-inflammatory cytokine secretion from multiple types of cells. To clarify whether de novo synthesized ceramide contributes to cytokine dysregulation in adipocytes and macrophages, we observed cytokine secretion in mature 3T3-L1 adipocytes (L1) and RAW264.7 macrophages (RAW) cultured alone or co-cultured under the suppression of de novo ceramide synthesis.Palmitate enhanced ceramide accumulation and stimulated the expression and secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in L1. The suppression of serine-palmitoyl transferase, a rate-limiting enzyme of de novo ceramide synthesis, by myriocin or siRNA attenuated those palmitate-induced alterations, and a ceramide synthase inhibitor fumonisin B1 showed similar results. In contrast, the inhibitor of sphingosine kinase or a membrane-permeable ceramide analogue augmented the cytokine secretion. Myriocin effects on the palmitate-induced changes were not abrogated by toll-like receptor-4 blockade. Although palmitate stimulated RAW to secrete tumor necrosis factor-α (TNF-α), it did not significantly increase ceramide content, and neither myriocin nor fumonisin B1 attenuated the TNF-α hypersecretion. The co-culture of L1 with RAW markedly augmented IL-6 and MCP-1 levels in media. Myriocin or fumonisin B1 significantly lowered these cytokine levels and suppressed the gene expression of TNF-α and MCP-1 in RAW and of IL-6 and MCP-1 in L1.In conclusion, de novo synthesized ceramide partially mediates the palmitate effects on pro-inflammatory adipokines and is possibly involved in the interaction with macrophages.     

Resistance of chimpanzee T cells to human immunodeficiency virus type 1 Tat-enhanced oxidative stress and apoptosis.

CD4+ T-cell depletion in AIDS patients involves induction of apoptosis in human immunodeficiency virus (HIV)-infected and noninfected T cells. The HIV type 1 (HIV-1)-transactivating protein Tat enhances apoptosis and activation-induced cell death (AICD) of human T cells. This effect is mediated by the CD95 (APO-1/Fas) receptor-CD95 ligand (CD95L) system and may be linked to the induction of oxidative stress by Tat. Here we show that HIV-1 Tat-induced oxidative stress is necessary for sensitized AICD in T cells caused by CD95L expression. Tat-enhanced apoptosis and CD95L expression in T cells are inhibited by neutralizing anti-Tat antibodies, antioxidants, and the Tat inhibitor Ro24-7429. Chimpanzees infected with HIV-1 show viral replication resembling early infection in humans but do not show T-cell depletion or progression towards AIDS. The cause for this discrepancy is unknown. Here we show that unlike Tat-treated T cells in humans, Tat-treated chimpanzee T cells do not show downregulation of manganese superoxide dismutase or signs of oxidative stress. Chimpanzee T cells are also resistant to Tat-enhanced apoptosis, AICD, and CD95L upregulation.     

Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.     

Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria

B-cells, triggered via their surface B-cell receptor (BcR), start an apoptotic program known as activation-induced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD, the contribution of either and the exact molecular events through which AICD commences are still unknown. Here we show that in Ramos B-cells, BcR-triggered cell death is associated with an early rise of C16 ceramide that derives from activation of the de novo pathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate. There was no evidence for activation of sphingomyelinases or hydrolysis of sphingomyelin. Importantly, FB1 inhibited several specific apoptotic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragmentation. Electron microscopy revealed morphological evidence of mitochondrial damage, suggesting the involvement of mitochondria in BcR-triggered apoptosis, and this was inhibited by FB1. Moreover, a loss of mitochondrial membrane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val-Ala-dl-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de novo generated C16 ceramide in the execution of AICD, and they further suggest an ordered and more specific sequence of biochemical events in which de novo generated C16 ceramide is involved in mitochondrial damage resulting in a downstream activation of caspases and apoptosis.     

Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein-coupled receptors or as an intracellular second messenger. Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide. Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.     

Entamoeba invadens: Sphingolipids metabolic regulation is the main component of a PKC signaling pathway in controlling cell growth and proliferation

The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917-924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6 h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4-8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous d-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.     

Ceramide synthase-dependent ceramide generation and programmed cell death: involvement of salvage pathway in regulating postmitochondrial events

The sphingolipid ceramide has been widely implicated in the regulation of programmed cell death or apoptosis. The accumulation of ceramide has been demonstrated in a wide variety of experimental models of apoptosis and in response to a myriad of stimuli and cellular stresses. However, the detailed mechanisms of its generation and regulatory role during apoptosis are poorly understood. We sought to determine the regulation and roles of ceramide production in a model of ultraviolet light-C (UV-C)-induced programmed cell death. We found that UV-C irradiation induces the accumulation of multiple sphingolipid species including ceramide, dihydroceramide, sphingomyelin, and hexosylceramide. Late ceramide generation was also found to be regulated by Bcl-xL, Bak, and caspases. Surprisingly, inhibition of de novo synthesis using myriocin or fumonisin B1 resulted in decreased overall cellular ceramide levels basally and in response to UV-C, but only fumonisin B1 inhibited cell death, suggesting the presence of a ceramide synthase (CerS)-dependent, sphingosine-derived pool of ceramide in regulating programmed cell death. We found that this pool did not regulate the mitochondrial pathway, but it did partially regulate activation of caspase-7 and, more importantly, was necessary for late plasma membrane permeabilization. Attempting to identify the CerS responsible for this effect, we found that combined knockdown of CerS5 and CerS6 was able to decrease long-chain ceramide accumulation and plasma membrane permeabilization. These data identify a novel role for CerS and the sphingosine salvage pathway in regulating membrane permeability in the execution phase of programmed cell death.     

An IL-2-dependent switch between CD95 signaling pathways sensitizes primary human T cells toward CD95-mediated activation-induced cell death

The CD95 (APO-1/Fas) system plays a critical role in activation-induced cell death (AICD) of T cells. We previously described two distinct CD95 (APO-1/Fas) signaling pathways: 1) type I cells show strong death-inducing signaling complex (DISC) formation and mitochondria-independent apoptosis and 2) DISC formation is reduced in type II cells, leading to mitochondria-dependent apoptosis. To investigate the relevance of these pathways, we set up an in vitro model that mimics the initiation and the down phase of an immune response, respectively. Freshly activated human T cells (initiation) are resistant toward CD95-mediated AICD despite high expression of CD95. We previously reported that these T cells show reduced DISC formation. In this study, we show that freshly activated T cells are CD95-type II cells that show high expression levels of Bcl-x(L) and display a block in the mitochondrial apoptosis pathway. Furthermore, we show that, upon prolonged culture (down phase), human T cells undergo a switch from type II to type I cells that renders T cells sensitive to CD95-mediated AICD. Finally, we demonstrate that this switch is dependent on the presence of IL-2. Our observations reveal for the first time that the existence of coexisting CD95 signaling pathways is of physiological relevance.     

Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1

The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) 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 B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.     

Novel role for mitochondria: protein kinase Ctheta-dependent oxidative signaling organelles in activation-induced T-cell death

Reactive oxygen species (ROS) play a key role in regulation of activation-induced T-cell death (AICD) by induction of CD95L expression. However, the molecular source and the signaling steps necessary for ROS production are largely unknown. Here, we show that the proximal T-cell receptor-signaling machinery, including ZAP70 (zeta chain-associated protein kinase 70), LAT (linker of activated T cells), SLP76 (SH2 domain-containing leukocyte protein of 76 kDa), PLCgamma1 (phospholipase Cgamma1), and PKCtheta (protein kinase Ctheta), are crucial for ROS production. PKCtheta is translocated to the mitochondria. By using cells depleted of mitochondrial DNA, we identified the mitochondria as the source of activation-induced ROS. Inhibition of mitochondrial electron transport complex I assembly by small interfering RNA (siRNA)-mediated knockdown of the chaperone NDUFAF1 resulted in a block of ROS production. Complex I-derived ROS are converted into a hydrogen peroxide signal by the mitochondrial superoxide dismutase. This signal is essential for CD95L expression, as inhibition of complex I assembly by NDUFAF1-specific siRNA prevents AICD. Similar results were obtained when metformin, an antidiabetic drug and mild complex I inhibitor, was used. Thus, we demonstrate for the first time that PKCtheta-dependent ROS generation by mitochondrial complex I is essential for AICD.     

Expression and biological activity of X-linked inhibitor of apoptosis (XIAP) in human malignant glioma.

The inhibitor-of-apoptosis (IAP) proteins are a novel family of antiapoptotic proteins that are thought to inhibit cell death via direct inhibition of caspases. Here, we report that human malignant glioma cell lines express XIAP, HIAP-1 and HIAP-2 mRNA and proteins. NAIP was not expressed. IAP proteins were not cleaved during CD95 ligand (CD95L)-induced apoptosis, and loss of IAP protein expression was not responsible for the potentiation of CD95L-induced apoptosis when protein synthesis was inhibited. LN-18 cells are highly sensitive to CD95-mediated apoptosis, whereas LN-229 cells require co-exposure to CD95L and a protein synthesis inhibitor, CHX, to acquire sensitivity to apoptosis. Adenoviral XIAP gene transfer blocked caspase 8 and 3 processing in both cell lines in the absence of CHX. Apoptosis was blocked in the absence and in the presence of CHX. However, XIAP failed to block caspase 8 processing in LN-229 cells in the presence of CHX. There was considerable overlap of the effects of XIAP on caspase processing with those of BCL-2 and the viral caspase inhibitor crm-A. These data define complex regulatory mechanisms for CD95-mediated apoptosis in glioma cells and indicate that there may be a distinct pathway of death receptor-mediated apoptosis that is readily activated when protein synthesis is inhibited. The constitutive expression of natural caspase inhibitors may play a role in the resistance of these cells to apoptotic stimuli that directly target caspases, including radiochemotherapy and immune-mediated tumor cell lysis.     

Fumonisins and fumonisin analogs as inhibitors of ceramide synthase and inducers of apoptosis

Sphingoid bases are growth inhibitory and pro-apoptotic for many types of cells when added to cells exogenously, and can be elevated to toxic amounts endogenously when cells are exposed to inhibitors of ceramide synthase. An important category of naturally occurring inhibitors are the fumonisins, which inhibit ceramide synthase through structural similarities with both the sphingoid base and fatty acyl-CoA co-substrates. Fumonisins cause a wide spectrum of disease (liver and renal toxicity and carcinogenesis, neurotoxicity, induction of pulmonary edema, and others), and most-possibly all-of the pathophysiologic effects of fumonisins are attributable to disruption of the sphingolipid metabolism. The products of alkaline hydrolysis of fumonisins (which occurs during the preparation of masa flour for tortillas) are aminopentols that also inhibit ceramide synthase, but more weakly. Nonetheless, the aminopentols (and other 1-deoxy analogs of sphinganine) are acylated to derivatives that inhibit ceramide synthase, perhaps as product analogs, elevate sphinganine, and kill the cells. Somewhat paradoxically, fumonisins sometimes stimulate growth and inhibit apoptosis, possibly due to elevation of sphinganine 1-phosphate, which is known to have these cellular effects. These findings underscore the complexity of sphingolipid metabolism and the difficulty of identifying the pertinent mediators unless a full profile of the potentially bioactive species is evaluated.     

Activation-induced cell death: the controversial role of Fas and Fas ligand in immune privilege and tumour counterattack

Activation-induced cell death (AICD) is the process by which cells undergo apoptosis in a controlled manner through the interaction of a death factor and its receptor. Programmed cell death can be induced by a number of physiological and pathological factors including Fas (CD95)-Fas ligand (FasL/CD95L) interaction, tumour necrosis factor (TNF), ceramide, and reactive oxygen species (ROS). Fas is a 48-kDa type I transmembrane protein that belongs to the TNF/nerve growth factor receptor superfamily. FasL is a 40-kDa type II transmembrane protein that belongs to the TNF superfamily. The interaction of Fas with FasL results in a series of signal transductions which initiate apoptosis. The induction of apoptosis in this manner is termed AICD. Activation-induced cell death and Fas-FasL interactions have been shown to play significant roles in immune system homeostasis. In this review the involvement of Fas and Fas ligand in cell death, with particular reference to the T cell, and the mechanism(s) by which they induce cell death is described. The role of AICD in immune system homeostasis and the controversy surrounding the role of FasL in immune privilege, inflammation, and so-called tumour counterattack is also discussed.     

Adhesion-mediated intracellular redistribution of c-Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein-long confers resistance to CD95-induced apoptosis in hematopoietic cancer cell lines

Evasion of immune surveillance is a key step in malignant progression. Interactions between transformed hematopoietic cells and their environment may initiate events that confer resistance to apoptosis and facilitate immune evasion. In this report, we demonstrate that beta(1) integrin-mediated adhesion to fibronectin inhibits CD95-induced caspase-8 activation and apoptosis in hematologic tumor cell lines. This adhesion-dependent inhibition of CD95-mediated apoptosis correlated with enhanced c-Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein-long (c-FLIP(L)) cytosolic solubility compared with nonadhered cells. Cytosolic c-FLIP(L) protein preferentially associated with cytosolic Fas-associated death domain protein (FADD) and localized to the death-inducing signal complex after CD95 ligation in adherent cells. The incorporation of c-FLIP(L) in the death-inducing signal complex prevented procaspase-8 processing and activation of the effector phase of apoptosis. Adhesion to fibronectin increased c-FLIP(L) cytosolic solubility and availability for FADD binding by redistributing c-FLIP(L) from a preexisting membrane-associated fraction. Increased cytosolic availability of c-FLIP(L) for FADD binding was not related to increased levels of RNA or protein synthesis. These data show that adhesion of anchorage-independent cells to fibronectin provides a novel mechanism of resistance to CD95-mediated programmed cell death by regulating the cellular localization and availability of c-FLIP(L).     

Aromatase inhibition by 15-deoxy-prostaglandin J(2) (15-dPGJ(2)) and N-(4-hydroxyphenyl)-retinamide (4HPR) is associated with enhanced ceramide production

Inhibition of aromatase activity is an established endocrine therapy in the treatment of hormone-dependent breast cancer. Recent studies on aromatase inhibition by the synthetic retinoid 4HPR, also known as fenretinide, and the PPARgamma agonist 15-dPGJ(2) have implicated a direct receptor-independent, redox-sensitive mechanism of action. The signalling molecule ceramide has also been previously implicated as a negative regulator of aromatase activity. In the present study, we have investigated a potential mediatory role for this sphingolipid during aromatase inhibition by fenretinide and 15-dPGJ(2) in the breast cancer cell line MDA MB 231 and JEG-3 choriocarcinoma cells. 4HPR and 15-dPGJ(2) caused a dose-dependent inhibition of aromatase activity associated with an increase in ceramide production. Both these actions were redox-sensitive as demonstrated by their abrogation in the presence of the anti-oxidant N-acetylcysteine. Exogenous ceramide analogue mimicked these inhibitory actions on aromatase, but in a redox-independent manner. Blockade of the de novo ceramide production pathway by fumonisin B(1) or myriocin inhibited the ceramide responses, but did not prevent aromatase inhibition by 15-dPGJ(2) or 4HPR. This study highlights a potential role for aromatase inhibition and the stress-response signal ceramide during the therapeutic actions of 15-dPGJ(2) and 4HPR in breast cancer treatment. However, these data do not support a mediatory role for this sphingolipid during aromatase inhibition by these agents.     

Synthesis of 4-hydroxysphinganine and characterization of sphinganine hydroxylase activity in corn

Sphinganine and 4-hydroxysphinganine (phytosphingosine) are the predominant free long-chain bases in lipid extracts of plant tissues. While the synthesis of sphinganine in plants has been investigated, the metabolic origin of 4-hydroxysphinganine is not known. Three different approaches utilizing fumonisin B(1), an inhibitor of sphinganine acylation, alone or in combination with beta-chloroalanine, an inhibitor of sphinganine synthesis, were used to establish that free 4-hydroxysphinganine is produced in excised corn shoots by the direct hydroxylation of sphinganine and not from the breakdown of complex sphingolipids. Sphinganine hydroxylase activity was characterized in microsomes isolated from corn. The enzyme was found to utilize D-erythro-sphinganine (with half-maximal activity observed at a substrate concentration of approximately 60 microM) and either NADPH (K(m)=33 microM) or NADH (K(m)=58 microM) as substrates. Ceramide hydroxylation was also demonstrated in corn microsomes, and the lack of competition between ceramide and sphinganine suggests the presence of distinct enzymes responsible for hydroxylating these two substrates. Using marker assays, sphinganine hydroxylase activity was localized to the endoplasmic reticulum. Sphinganine hydroxylase activity in microsomes isolated from corn shoots treated with fumonisin B(1) increased more than 3-fold compared to controls. The results of this study shed light on sphingolipid long-chain base synthesis and modification in plant tissues and suggest a possible contribution of sphinganine hydroxylase in manifesting the effects of fumonisin in plants.     

Fatty acid protection from palmitic acid-induced apoptosis is lost following PI3-kinase inhibition

We have previously shown that saturated fatty acids induce DNA damage and cause apoptotic cell death in insulin-producing beta-cells. Here we examine further the effects of single or combined dietary fatty acids on RINm5F survival or cell death signalling. Palmitate and stearate, but not linoleate, oleate or palmitoylmethyl ester, induced growth inhibition and increased apoptosis in RINm5F cells following 24 h exposure. Co-incubation with inhibitors of ceramide synthesis, myriocin or fumonisin B(1), did not improve viability of palmitic acid treated RINm5F cells. The inhibitor of inducible nitric oxide synthase, 1400 W, similarly had no protective effect. However, linoleic acid protected against palmitic acid-induced apoptotic and necrotic cell death. The specific pharmacological inhibitors of phosphatidylinositol 3-kinase, LY294002 and wortmannin, abolished the protective effect of linoleic acid on apoptosis but not on necrosis. These data show that the growth inhibitory and apoptosis-inducing effect of the saturated fatty acid palmitate on RINm5F cells is prevented by co-incubation with the polyunsaturated fatty acid linoleate but not inhibitors of ceramide or nitric oxide generation. A key role for phosphatidylinositol 3-kinase in mediating the linoleic-acid reduction in apoptosis is suggested.