Biomedical subjects

B Ségui

Publications and source records attributed to B Ségui.

11 recordsLinked to original sources

PubMed2006

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

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

Caspases↗

PubMed2001

The CB(1) cannabinoid receptor of astrocytes is coupled to sphingomyelin hydrolysis through the adaptor protein fan.

Cannabinoids exert most of their effects through the CB(1) receptor. This G protein-coupled receptor signals inhibition of adenylyl cyclase, modulation of ion channels, and stimulation of mitogen- and stress-activated protein kinases. In this article, we report that Delta(9)-tetrahydrocannabinol (THC), the major active component of marijuana, induces sphingomyelin hydrolysis in primary astrocytes but not in other cells expressing the CB(1) receptor, such as primary neurons, U373 MG astrocytoma cells, and Chinese hamster ovary cells transfected with the CB(1) receptor cDNA. THC-evoked sphingomyelin breakdown in astrocytes was also exerted by the endogenous cannabinoid anandamide and the synthetic cannabinoid HU-210 and was prevented by the selective CB(1) antagonist SR141716. By contrast, the effect of THC was not blocked by pertussis toxin, pointing to a lack of involvement of G(i/o) proteins. A role for the adaptor protein FAN in CB(1) receptor-coupled sphingomyelin breakdown is supported by two observations: 1) coimmunoprecipitation experiments show that the binding of FAN to the CB(1) receptor is enhanced by THC and prevented by SR141716; 2) cells expressing a dominant-negative form of FAN are refractory to THC-induced sphingomyelin breakdown. This is the first report showing that a G-protein-coupled receptor induces sphingomyelin hydrolysis through FAN and that the CB(1) cannabinoid receptor may signal independently of G(i/o) proteins.

Adenylate Cyclase Toxin↗

PubMed2001

Involvement of FAN in TNF-induced apoptosis.

TNF-alpha is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.

Amino Acid Chloromethyl Ketones↗

PubMed2000

Lysosomal sphingomyelinase is not solicited for apoptosis signaling.

Stress-induced activation of an acidic sphingomyelinase leading to generation of ceramide, an important lipid mediator, has been associated with apoptosis; however, the implication of this hydrolase has been questioned. The present study aimed at re-evaluating the role of this lysosomal enzyme in apoptosis initiated by different apoptotic inducers. The sensitivity of a series of acid sphingomyelinase-deficient cell lines derived from Niemann-Pick disease patients to stress-induced apoptosis was investigated. We have now shown that stress stimuli, such as anthracyclines, ionizing radiation, and Fas ligation trigger similar apoptotic hallmarks in normal and acid sphingomyelinase-deficient cell lines. Retrovirus-mediated gene correction of enzyme deficiency in Niemann-Pick cells does not modify response to apoptosis. Ceramide production is comparable in normal and Niemann-Pick cells, and increased activity of neutral sphingomyelinase is observed. Thus, our findings cast serious doubts that lysosomal sphingomyelinase activation is responsible for stress-induced apoptosis of cultured cells.

Apoptosis↗

PubMed2000

Stress-induced apoptosis is not mediated by endolysosomal ceramide.

A major lipid-signaling pathway in mammalian cells implicates the generation of ceramide from the ubiquitous sphingolipid sphingomyelin (SM). Hydrolysis of SM by a sphingomyelinase present in acidic compartments has been reported to mediate, via the production of ceramide, the apoptotic cell death triggered by stress-inducing agents. In the present study, we investigated whether the ceramide formed within or accumulated in lysosomes indeed triggers apoptosis. A series of observations strongly suggests that ceramide involved in stress-induced apoptosis is not endolysosomal: 1) Although short-chain ceramides induced apoptosis, loading cells with natural ceramide through receptor-mediated endocytosis did not result in cell death. 2) Neither TNF-alpha nor anti-CD95 induced the degradation to ceramide of a natural SM that had been first introduced selectively into acidic compartments. 3) Stimulation of SV40-transformed fibroblasts by TNF-alpha or CD40 ligand resulted in apoptosis equally well in cells derived from control individuals and from patients affected with Farber disease, having a genetic defect of acid ceramidase activity leading to lysosomal accumulation of ceramide. Also, induction of apoptosis using anti-CD95 (Fas) or anti-CD40 antibodies, TNF-alpha, daunorubicin, and ionizing radiation was similar in control and Farber disease lymphoid cells. In all cases, apoptosis was preceded by a comparable increase of intracellular ceramide levels. 4) Retroviral-mediated gene transfer and overexpression of acid ceramidase in Farber fibroblasts, which led to complete metabolic correction of the ceramide catabolic defect, did not affect the cell response to TNF-alpha and CD40 ligand.

Apoptosis↗

PubMed1999

CD40 signals apoptosis through FAN-regulated activation of the sphingomyelin-ceramide pathway.

The possibility that the sphingomyelin (SM)-ceramide pathway is activated by CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily and that plays a critical role in the regulation of immune responses has been investigated. We demonstrate that incubation of Epstein-Barr virus-transformed lymphoid cells with an anti-CD40 antibody acting as an agonist results in the stimulation of a neutral sphingomyelinase, hydrolysis of cellular SM, and concomitant ceramide generation. In addition, SM degradation was observed in acid sphingomyelinase-deficient cells, as well as after ligation by soluble CD40 ligand. The anti-CD40 antibody, as well as the soluble CD40 ligand induced a decrease in thymidine incorporation and morphological features of apoptosis, which were mimicked by cell-permeant or bacterial sphingomyelinase-produced ceramides. Stable expression of a dominant-negative form of the FAN protein (factor associated with neutral sphingomyelinase activation), which has been reported to mediate tumor necrosis factor-induced activation of neutral sphingomyelinase, significantly inhibited CD40 ligand-induced sphingomyelinase stimulation and apoptosis of transformed human fibroblasts. Transformed fibroblasts from FAN knockout mice were also protected from CD40-mediated cell death. Finally, anti-CD40 antibodies were able to co-immunoprecipitate FAN in control fibroblasts but not in cells expressing the dominant-negative form of FAN, indicating interaction between CD40 and FAN. Altogether, these results strongly suggest that CD40 ligation can activate via FAN a neutral sphingomyelinase-mediated ceramide pathway that is involved in the cell growth inhibitory effects of CD40.

Animals↗

PubMed1999

Sphingomyelin-degrading pathways in human cells role in cell signalling.

The ubiquitous sphingophospholipid sphingomyelin (SM) can be hydrolysed in human cells to ceramide by different sphingomyelinases (SMases). These enzymes exert a dual role, enabling not only the turnover of membrane SM and the degradation of exogenous (lipoprotein) SM, but also the signal-induced generation of the lipid second messenger ceramide. This review focuses on the function(s) of the different SMases in living cells. While both lysosomal and non-lysosomal pathways that ensure SM hydrolysis in intact cells can be distinguished, the precise contribution of each of these SM-cleaving enzymes to the production of ceramide as a signalling molecule remains to be clarified.

Animals↗

PubMed1998

Evidence for the lack of involvement of sphingomyelin hydrolysis in the tumor necrosis factor-induced secretion of nerve growth factor in primary astrocyte cultures.

The signal mechanism underlying tumor necrosis factor alpha (TNF alpha) up-regulation of nerve growth factor (NGF) production was studied in primary rat astrocyte cultures. Because ceramide is also able to induce NGF secretion and because TNF alpha is a known agonist of the sphingomyelin (SPM)-ceramide pathway, we investigated whether the TNF alpha-induced NGF secretion by primary astrocytes is mediated by ceramide. TNF alpha stimulation of NGF secretion was shown to be independent of protein kinase C, abrogated by the tyrosine phosphoprotein phosphatase inhibitor phenylarsine oxide (PAO), and independent of the activation of the mitogen-activated protein kinase (MAPK) cascade. In marked contrast, inhibition of MAPK counteracted the NGF secretion induced by ceramide. TNF alpha stimulation of the nuclear transcription factor NF-kappaB was prevented by cell pretreatment with PAO, whereas ceramide and sphingomyelinase had a marginal effect on NF-kappaB activation. Moreover, TNF alpha failed to activate the SPM pathway, as indicated by the lack of SPM degradation and the absence of ceramide generation. To clarify further the role of NF-kappaB in NGF synthesis, electrophoretic mobility shift assays were performed with an NF-kappaB site from the NGF promoter. The absence of significant binding of NF-kappaB to the NGF gene promoter indicates the existence of an indirect role of NF-kappaB in the regulation of NGF synthesis. Altogether, our data strongly suggest that TNF alpha-mediated up-regulation of NGF occurs independently of ceramide generation.

Animals↗

PubMedDate not supplied

Use of human mesenteric arteries to study chronic vascular rejection in SCID/beige mice reconstituted with human spleen cells.

We wanted to establish a preclinical model of chronic vascular rejection (CVR) by transplanting small arteries from the mesentery of cadaveric organ donors by the rapid "sleeve" technique into SCID/beige mice reconstituted with human allogeneic spleen cells. After institutional authorization and with informed consent from relatives, we obtained tissues and cells from cadaveric organ donors. A piece of mesentery was recovered from the donor and kept in buffered solution at 4 degrees C until use. After dissection of the mesentery, small arteries of suitable size were transplanted in place of the infrarenal aorta of the mice. Cells for the immunological reconstitution of the mice were spleen cells from the same or other organ donors. Twenty-three suitable arterial segments were obtained from the mesentery of three cadaveric donors. Ten of the mice received 3 x 10(7) human spleen cells intraperitoneally 1 week after the arterial graft and they all showed circulating human CD3+ and CD19+ cells 2 weeks after injection. The mice were sacrificed 5 weeks after the arterial graft. SCID/beige mice reconstituted with allogeneic spleen cells showed a typical CVR, whereas mice that received no cells had a normal vascular anatomy. We believe our model is well suited for the study of treatment of CVR under human allograft conditions.

Animals↗

PubMedDate not supplied

Chronic vascular rejection: histologic comparison between two murine experimental models.

BACKGROUND: We previously developed an experimental model to study chronic vascular rejection (CVR) in mice, the orthotopic aortic allograft. More recently we performed human arterial grafts into SCID/Beige mice reconstituted with human spleen cells. We report herein the differences in CVR lesions. MATERIAL AND METHODS: In the first model, recipient mice were C57BL/6 (H-2b), and donor mice were DBA/2 (H-2d). In the second model, terminal branches of the human superior mesenteric artery were transplanted into SCID/Beige mice in the infrarenal aorta. Human immune reconstitution was achieved by a single intraperitoneal injection of 30 x 10(6) human spleen cells. The presence of human lymphocytes and IgG was verified weekly. In both models, the vascular grafts were inserted in the infrarenal aortic position using the sleeve technique. The transplanted mice were sacrificed at 35 days after the operation. The grafts were analyzed by histology and morphometry. The mean intimal thickening was calculated based on transverse sections at 0.1-mm intervals. RESULTS: Typical CVR lesions developed with neointimal thickening, T-cell infiltration, and smooth muscle cell (SMC) proliferation in both models. In the mouse aortic model, disappearance of SMC in the media was noted in contrast to human arterial transplants, where the media remained intact. CONCLUSION: Other groups have noted that arteries conserve their media in clinical organ transplants. From this point of view, the lesions in the second experimental model (human arteries) better reflect the pathology of CVR in clinical transplantation than the murine aortic transplant model.

Animals↗

PubMedDate not supplied

Human immune reconstitution with spleen cells in SCID/Beige mice.

BACKGROUND: We developed an original experimental model to study chronic vascular rejection (CVR) consisting of a graft of human mesenteric artery followed by human immune reconstitution into CB.17 SCID/Beige mice. Human immune reconstitution achieved after human PBMC injection has often been variable and incomplete. The aim of this work was to develop an alternative method to achieve a complete, functional human immune reconstitution. METHOD: After institutional authorizations, spleen cells were recovered from cadaveric organ donors. Single intraperitoneal injections of various doses of spleen cells were made into 70 CB.17 SCID/Beige mice. Reconstitution of the human immune system was monitored by flow cytometry (circulating human cells) and ELISA (human IgG). Colonization of murine lymphoid organs by human cells was studied by immunohistochemistry and flow cytometry. Evaluation of the immune function consisted of examination of CVR lesions in human arterial grafts. The animals were humanely killed at day 28. RESULTS: After injection of 30 to 40 x 10(6) spleen cells, the mice showed significant human CD3(+), CD19(+), and CD56(+) populations in peripheral blood. The mean human cells levels were, respectively, 8.2% +/- 5.4%, 2.9% +/- 1.2%, and 5.3% +/- 5.1%. Murine spleen and mesenteric lymph nodes were colonized by human T and B cells, while the murine thymus was only colonized by human T cells. Human IgG was detected in murine serum (65.9 +/- 63.3 mg/L) and typical CVR lesions were observed within the allogeneic grafts. CONCLUSION: Intraperitoneal injection of 30 to 40 x 10(6) human spleen cells into CB.17 SCID/Beige mice induces complete and functional human immune reconstitution allowing the study of CVR under human allogeneic conditions.

Aged↗