Reactive oxygen species generation is independent of de novo sphingolipids in apoptotic photosensitized cells

Our recent studies have shown that the de novo sphingolipids play a role in apoptosis of photosensitized cells. To elucidate the involvement of the de novo sphingolipids in reactive oxygen species (ROS) production and mitochondrial depolarization during apoptosis, the stress inducer photodynamic therapy (PDT) with the photosensitizer Pc 4 was used. In Jurkat cells PDT-triggered ROS production or mitochondrial membrane potential (deltapsi(m)) loss was not prevented by the de novo sphingolipid synthesis inhibitor ISP-1. However, PDT + C16-ceramide led to enhanced mitochondrial depolarization and DEVDase activation. The superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) protected Jurkat cells from ROS generation and apoptosis, but not from deltapsi(m) reduction. Sphinganine or C16-ceramide counteracted MnTBAP-induced protection from apoptosis in Jurkat, as well as CHO cells. In LY-B cells, CHO-derived mutants deficient in serine palmitoyltransferase (SPT) activity and the de novo sphingolipid synthesis, mitochondrial depolarization, but not ROS generation, was suppressed post-PDT. In LY-B cells transfected with the SPT component LCB1, deltapsi(m) collapse post-PDT was restored. The data support the following hypotheses: MnTBAP protects against apoptosis via steps downstream of deltapsi(m) loss; de novo sphingolipids are not required for ROS generation, but can play a role in deltapsi(m) dissipation in photosensitized apoptotic cells.     

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.     

Enhanced phagocytosis through inhibition of de novo ceramide synthesis

Fcgamma receptors are important mediators of the binding of IgG to and induction of phagocytosis in neutrophils. COS-1 cells provide a potentially useful model for studying these receptors because transfection with the FcgammaRIIA renders these cells phagocytic. During FcgammaRIIA-mediated phagocytosis in COS-1 cells, endogenous ceramide levels increased 52% by 20 min (p < 0.01). Phospholipase D activity increased by 62% (p < 0.01). Correspondingly, the phagocytic index increased by 3.7-fold by 20 min. Two inhibitors of ceramide formation were used to assess the consequences of reduced ceramide generation. l-Cycloserine, an inhibitor that blocks serine palmitoyltransferase activity, lowered both sphingosine and ceramide levels. Under these conditions, the phagocytic index increased 100% in the presence of 2 mm l-cycloserine. The formation of ceramide resulting from the N-acylation of dihydrosphingosine or sphingosine by ceramide synthase is inhibited by the fungal toxin fumonisin B(1). When cells were treated with 5-50 microm fumonisin B(1), the cellular level of ceramide decreased in a concentration-dependent manner, while simultaneously the phagocytic index increased by 52%. Concomitantly, three indirect measures of FcgammaRIIA activity were altered with the fall in ceramide levels. Syk phosphorylation, phospholipase D activity, and mitogen-activated protein (MAP) kinase phosphorylation were increased at 30 min. When Syk phosphorylation was blocked with piceatannol and cells were similarly challenged, phosphatidylinositol 3-kinase activation was blocked, but no changes in either ceramide accumulation or MAP kinase activation were observed. Ceramide formation and MAP kinase activation are therefore not dependent on Syk kinase activity in this system. These results indicate that COS-1 cells provide a useful model for the recapitulation of sphingolipid signaling in the study of phagocytosis. Ceramide formed by de novo synthesis may represent an important mechanism in the regulation of phagocytosis.     

Suramin induced ceramide accumulation leads to apoptotic cell death in dorsal root ganglion neurons

Suramin is an experimental antineoplastic agent that is currently being tested in clinical trials for a number of human cancers. In previous clinical trials, it has been noted that a significant percentage of patients treated with suramin develop a peripheral neuropathy. Both the cytotoxic (chemotherapeutic) and neurotoxic mechanisms of action of this compound are unknown. Evidence presented in this study suggests that both effects may be due to extensive disruption in glycolipid transport and/or metabolism. Suramin treated dorsal root ganglion cultures revealed an accumulation of the GM1 ganglioside and ceramide. Exposure of cultures to suramin, a cell permeable ceramide analog, or sphingomyelinase lead to apoptotic cell death demonstrated by electron microscopy, bis-benzimide staining and DNA laddering on gel electrophoresis. Furthermore, a significant increase in intracellular ceramide preceded cell death in suramin treated neurons. We propose that suramin induced ceramide accumulation within neurons leads to apoptotic cell death.     

Male germ cells require polyenoic sphingolipids with complex glycosylation for completion of meiosis: a link to ceramide synthase-3

Previously, it was found that a novel class of neutral fucosylated glycosphingolipids (GSLs) is required for male fertility. These lipids contain very long-chain (C26-C32) polyunsaturated (4-6 double bonds) fatty acid residues (VLC-PUFAs). To assess the role of these complex GSLs in spermatogenesis, we have now investigated with which of the testicular cell types these lipids are associated. During postnatal development, complex glycosylated and simple VLC-PUFA sphingolipids were first detectable at day 15, when the most advanced germ cells are pachytene spermatocytes. Their synthesis is most likely driven by ceramide synthase-3. This enzyme is encoded by the Cers3/Lass3 gene (longevity assurance genes), and out of six members of this gene family, only Cers3 mRNA expression was limited to germ cells, where it was up-regulated more than 700-fold during postnatal testicular maturation. Increasing levels of neutral complex VLC-PUFA GSLs also correlated with the progression of spermatogenesis in a series of male sterile mutants with arrests at different stages of spermatogenesis. Remarkably, fucosylation of the complex VLC-PUFA GSLs was not essential for spermatogenesis, as fucosylation-deficient mice produced nonfucosylated versions of the complex testicular VLC-PUFA GSLs, had complete spermatogenesis, and were fertile. Nevertheless, sterile Galgt1(-/-) mice, with a defective meiotic cytokinesis and a subsequent block in spermiogenesis, lacked complex but contained simple VLC-PUFA GSLs, as well as VLC-PUFA ceramides and sphingomyelins, indicating that the latter lipids are not sufficient for completion of spermatogenesis. Thus, our data imply that both glycans and the particular acyl chains of germinal sphingolipids are relevant for proper completion of meiosis.     

Serine palmitoyltransferase: role in apoptotic de novo ceramide synthesis and other stress responses

Serine palmitoyltransferase is the first and rate-limiting enzyme of sphingolipid synthesis. As such, it is a central control point in the synthesis of bioactivate sphingolipids, and it plays an important role in mediating cellular stress responses. In this review, its role in mediating these responses is discussed within the context of de novo ceramide synthesis. Furthermore, a discussion is provided of its regulation as discerned from both yeast and mammalian studies.     

Yeast cells lacking all known ceramide synthases continue to make complex sphingolipids and to incorporate ceramides into glycosylphosphatidylinositol (GPI) anchors

In yeast, the inositolphosphorylceramides mostly contain C26:0 fatty acids. Inositolphosphorylceramides were considered to be important for viability because the inositolphosphorylceramide synthase AUR1 is essential. However, lcb1Δ cells, unable to make sphingoid bases and inositolphosphorylceramides, are viable if they harbor SLC1-1, a gain of function mutation in the 1-acyl-glycerol-3-phosphate acyltransferase SLC1. SLC1-1 allows the incorporation of C26:0 fatty acids into phosphatidylinositol (PI), thus generating PI″, an abnormal, C26-containing PI, presumably acting as surrogate for inositolphosphorylceramide. Here we show that the lethality of the simultaneous deletion of the known ceramide synthases LAG1/LAC1/LIP1 and YPC1/YDC1 can be rescued by the expression of SLC1-1 or the overexpression of AUR1. Moreover, lag1Δ lac1Δ ypc1Δ ydc1Δ (4Δ) quadruple mutants have been reported to be viable in certain genetic backgrounds but to still make some abnormal uncharacterized inositol-containing sphingolipids. Indeed, we find that 4Δ quadruple mutants make substantial amounts of unphysiological inositolphosphorylphytosphingosines but that they also still make small amounts of normal inositolphosphorylceramides. Moreover, 4Δ strains incorporate exogenously added sphingoid bases into inositolphosphorylceramides, indicating that these cells still possess an unknown pathway allowing the synthesis of ceramides. 4Δ cells also still add quite normal amounts of ceramides to glycosylphosphatidylinositol anchors. Synthesis of inositolphosphorylceramides and inositolphosphorylphytosphingosines is operated by Aur1p and is essential for growth of all 4Δ cells unless they contain SLC1-1. PI″, however, is made without the help of Aur1p. Furthermore, mannosylation of PI″ is required for the survival of sphingolipid-deficient strains, which depend on SLC1-1. In contrast to lcb1Δ SLC1-1, 4Δ SLC1-1 cells grow at 37 °C but remain thermosensitive at 44 °C.     

Curcumin induces apoptosis of multidrug-resistant human leukemia HL60 cells by complex pathways leading to ceramide accumulation

Most anti-cancer agents induce apoptosis, however, a development of multidrug resistance in cancer cells and defects in apoptosis contribute often to treatment failure. Here, the mechanism of curcumin-induced apoptosis was investigated in human leukemia HL60 cells and their HL60/VCR multidrug-resistant counterparts. In both cell lines curcumin induced a bi-phasic ceramide generation with a slow phase until 6 h followed by a more rapid one. The level of the ceramide accumulation correlated inversely with the cell viability. We found that the ceramide elevation resulted from multifarious changes of the activity of sphingolipid-modifying enzymes. In both cell lines curcumin induced relatively fast activation of neutral sphingomyelinase (nSMase), which peaked at 3 h, and was followed by inhibition of sphingomyelin synthase activity. In addition, in HL60/VCR cells the glucosylceramide synthase activity was diminished by curcumin. This process was probably due to curcumin-induced down-regulation of P-gp drug transporter, since cyclosporine A, a P-gp blocker, also inhibited the glucosylceramide synthase activity. Inhibition of nSMase activity with GW4869 or silencing of SMPD3 gene encoding nSMase2 reversed the curcumin-induced inhibition of sphingomyelin synthase without affecting the glucosylceramide synthase activity. The early ceramide generation by nSMase was indispensable for the later lipid accumulation, modulation of Bax, Bcl-2 and caspase 3 levels, and for reduction of cell viability in curcumin-treated cells, as all these events were inhibited by GW4869 or nSMase2 depletion. These data indicate that the early ceramide generation by nSMase2 induced by curcumin intensifies the later ceramide accumulation via inhibition of sphingomyelin synthase, and controls pro-apoptotic signaling.     

De novo production of antigen-specific suppressor cells in vivo

Foxp3-expressing regulatory T cells (Treg) play an essential role in maintaining tolerance to self antigens and are generated under physiological conditions when developing T cells encounter antigens expressed by thymic epithelial cells. We have addressed the possibility that Treg can be exploited to prevent or even suppress ongoing immune responses to foreign antigens. To this end, one must develop methods that permit the de novo generation of Treg specific for foreign antigens in peripheral lymphoid tissue. This report describes the methodology of generating Treg by delivering minute doses of peptide contained in fusion Abs directed against the DEC-205 endocytic receptor on steady-state dendritic cells. The process, from cloning and production of fusion Abs to antigen-specific Treg induction in vivo, takes approximately 2 months. The results show that delivery of T-cell receptor agonist ligands under subimmunogenic conditions represents a suitable approach for converting naive T cells into Treg.     

Synthetic, non-natural sphingolipid analogs inhibit the biosynthesis of cellular sphingolipids, elevate ceramide and induce apoptotic cell death

Numerous studies have demonstrated the participation of sphingolipids in signal transduction and regulation of cell growth. Several cellular stress agents have been shown to elevate ceramide, the basic precursor of all sphingolipids, initiating a cascade of events leading to arrest of the cell cycle, apoptosis and cell death. Aiming at inhibiting metabolic pathways of sphingolipid metabolism that might lead to an increase of cellular ceramide, we have synthesized non-natural analogs of ceramide, sphingosine and trimethylsphingosine. When the respective analogs were applied to HL60 human myeloid leukemic cells they inhibited the biosynthesis of sphingomyelin (SPM) and glycosphingolipids and induced apoptosis that led to cell death. A fluorescent procedure which has been developed for quantifying the biosynthesis of cellular ceramide indicated an increase in the ceramide content following an incubation with the synthetic analogs. These results suggest that the newly synthesized sphingolipid analogs might be valuable for potential application as a therapeutic modality in leukemia and other malignancies.     

Regulation of insulin action by ceramide: dual mechanisms linking ceramide accumulation to the inhibition of Akt/protein kinase B

The sphingolipid ceramide negatively regulates insulin action by inhibiting Akt/protein kinase B (PKB), a serine/threonine kinase that is a central regulator of glucose uptake and anabolic metabolism. Despite considerable attention, the molecular mechanism accounting for this action of ceramide has remained both elusive and controversial. Herein we utilized deletion constructs encoding two different functional domains of Akt/PKB to identify which region of the enzyme conferred responsiveness to ceramide. Surprisingly the findings obtained with these separate domains reveal that ceramide blocks insulin stimulation of Akt/PKB by two independent mechanisms. First, using the isolated pleckstrin homology domain, we found that ceramide specifically blocks the translocation of Akt/PKB, but not its upstream activator phosphoinositide-dependent kinase-1, to the plasma membrane. Second, using a construct lacking this pleckstrin homology domain, which does not require translocation for activation, we found that ceramide stimulates the dephosphorylation of Akt/PKB by protein phosphatase 2A. Collectively these findings identify at least two independent mechanisms by which excessive ceramide accumulation in peripheral tissues could contribute to the development of insulin resistance. Moreover the results obtained provide a unifying theory to account for the numerous dissenting reports investigating the actions of ceramide toward Akt/PKB.     

De novo generation of a PrPSc-like conformation in living cells

Conformational conversion of the cellular PrPC protein to PrPSc is a central aspect of the prion diseases, but how PrP initially converts to this conformation remains a mystery. Here we show that PrP expressed in the yeast cytoplasm, instead of the endoplasmic reticulum, acquires the characteristics of PrPSc, namely detergent insolubility and a distinct pattern of protease resistance. Neuroblastoma cells cultured under reducing, glycosylation-inhibiting conditions produce PrP with the same characteristics. We therefore describe what is, to our knowledge, the first conversion of full-length PrP in a heterologous system, show the importance of reducing and deglycosylation conditions in PrP conformational transitions, and suggest a model for initiating events in sporadic and inherited prion diseases.     

De novo complex chromosome rearrangement: a study of two patients

Complex chromosome rearrangements (CCR) involving multiple breaks in two or more chromosomes are rare. We describe a girl with development delay and overgrowth who presents a nine-break apparently balanced de novo rearrangement involving chromosomes 1, 2, 3, 4 and 12, and a boy with developmental delay and seizures with a complex three-chromosome apparently balanced de novo rearrangement involving chromosomes 2, 7 and 13. The relationship between clinical abnormalities and apparently balanced rearrangements is discussed.     

Hyperresistance to photosensitized lipid peroxidation and apoptotic killing in 5-aminolevulinate-treated tumor cells overexpressing mitochondrial GPX4

Antitumor photodynamic therapy (PDT) with administered 5-aminolevulinic acid (ALA) is based on metabolism of ALA to protoporphyrin IX (PpIX), which acts as a sensitizer of photo-oxidative damage leading to apoptotic or necrotic cell death. An initial goal of this study was to ascertain how the PpIX-sensitized death mechanism for a breast tumor line (COH-BR1 cells) might be influenced by the conditions of ALA exposure in vitro. Two different treatment protocols were developed for addressing this question: (i) continuous incubation with 1 mM ALA for 90 min; and, (ii) discontinuous incubation, i.e., 15 min with 1 mM ALA followed by 225 min without it. Following exposure to 2 J/cm2 of visible light, cell viability, death mechanism, and lipid hydroperoxide (LOOH) level were evaluated for each protocol using thiazolyl blue, Hoechst staining, and HPLC with electrochemical detection assays, respectively. PpIX was found to sensitize apoptosis when it existed mainly in mitochondria (protocol-1), but necrosis when it diffused to other sites, including plasma membrane (protocol-2). Experiments with a transfectant clone, 7G4, exhibiting approximately 85 times greater activity of the LOOH-detoxifying selenoenzyme GPX4 than parental cells, provided additional information about death mechanism. Located predominantly in mitochondria of 7G4 cells, GPX4 strongly inhibited both LOOH accumulation and apoptosis under protocol-1 conditions, but had no significant effect under protocol-2 conditions. These findings support the hypothesis that LOOHs produced by attack of photogenerated singlet oxygen on mitochondrial membrane lipids play an important early role in the apoptotic death cascade.     

De novo ceramide synthesis is responsible for the anti-tumor properties of camptothecin and doxorubicin in follicular thyroid carcinoma

Doxorubicin and camptothecin are two cytotoxic chemotherapeutic agents triggering apoptosis in various cancer cells, including thyroid carcinoma cells. Recent studies revealed a critical role of ceramide in chemotherapy and suggested that anti-cancer drugs may kill tumor cells through sphingomyelinase activation. However, in comparison to sphingomyelin hydrolysis, the relative involvement of de novo ceramide synthesis remained poorly explored and highly controversial. Here, we evidenced that both doxorubicin and camptothecin triggered ceramide accumulation in thyroid carcinoma cells. We demonstrated that ceramide increase occurred via the de novo pathway without neither acidic nor neutral sphingomyelinase contribution. Interestingly, de novo ceramide generation was responsible for the drug-induced malignant cell apoptosis through a caspase-3-dependent pathway and a decrease of thrombospondin amount. Furthermore, blocking ceramide metabolism by inhibiting glucosylceramide synthase strengthened the camptothecin and doxorubicin-dependent effects. Altogether, we evidenced that de novo ceramide synthesis mediates the anti-tumor properties of doxorubicin and camptothecin in thyroid carcinoma and suggested that glucosylation of ceramide may contribute to the drug-resistance phenotype in thyroid malignancies.     

De novo complex chromosome rearrangement in identical twins with multiple congenital anomalies

Summary A de novo and apparently balanced complex chromosome rearrangement (CCR) was found in monozygotic (MZ) twin infants with multiple congenital anomalies. The rearrangement involved 4 chromosomes with 6 breakpoints including 2p23, 2q13, 2q21.1, 3p23, 11q13.1, and 12q24.1. This seems to be the first report of a CCR in MZ twins. The relationship between this chromosome abnormality and MZ twinning is discussed.     

Relationships of apoptotic signaling mediated by ceramide and TNF-alpha in U937 cells

It is commonly assumed that ceramide is a second messenger that transduces signaling leading to apoptosis. We tested this hypothesis by investigating the role of ceramide in TNF-alpha-initiated apoptotic signaling using the histiocytic lymphoma cell line U937. We found considerable differences between cell killing by TNF-alpha and by ceramide. U937 cells treated with TNF-alpha are committed early and irreversibly to the apoptotic pathway and start to die 90 min after treatment. U937 cells treated with ceramide start to die 12 h after the initial treatment. The cell death signaling initiated by TNF-alpha is transduced within minutes of exposure to TNF-alpha and it is irreversible. Exogenous ceramide increases the intracellular level of ceramide rapidly, significantly, and well above the physiological levels, within minutes, but cellular commitment to death does not occur until after the first 6 h of incubation. Furthermore, the endogenous ceramide in U937 cells treated with TNF-alpha increases well after the commitment to the apoptotic pathway. The differences between ceramide and TNF-alpha in the kinetics and the commitment to the apoptotic pathway suggest that, (a) ceramide is not a second messenger in the apoptotic signaling of TNF-alpha, (b) ceramide elevations, in TNF-alpha treated cells, are a consequence rather than a cause of apoptosis and (c) exogenously added ceramide and TNF-alpha kill cells via different pathways.     

Development of a new doubly-labeled fluorescent ceramide probe for monitoring the metabolism of sphingolipids in living cells

A new ceramide analog, 1, containing two fluorescent dyes, NBD in the N-acyl part and KFL5 in the alkyl part, was synthesized. The fluorescence from both NBD and KFL5 was detected in living cells in a time-dependent manner. A multi-wavelength fluorescence detector was used to detect ceramide metabolites including sphingosine, sphingosine-1-phosphate, glucosylceramide, and sphingomyelin, which are connected to the fluorescent dyes, simultaneously in a single TLC plate.