Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis.

We report here that anticancer drugs such as doxorubicin lead to induction of the CD95 (APO-1/Fas) system of apoptosis and the cellular stress pathway which includes JNK/SAPKs. Ceramide, which accumulates in response to different types of cellular stress such as chemo- and radiotherapy, strongly induced expression of CD95-L, cleavage of caspases and apoptosis. Antisense CD95-L as well as dominant-negative FADD inhibited ceramide- and cellular stress-induced apoptosis. Fibroblasts from type A Niemann-Pick patients (NPA), genetically deficient in ceramide synthesis, failed to up-regulate CD95-L expression and to undergo apoptosis after gamma-irradiation or doxorubicin treatment. In contrast, JNK/SAPK activity was still inducible by doxorubicin in the NPA cells, suggesting that activation of JNK/SAPKs alone is not sufficient for induction of the CD95 system and apoptosis. CD95-L expression and apoptosis in NPA fibroblasts were restorable by exogenously added ceramide. In addition, NPA fibroblasts undergo apoptosis after triggering of CD95 with an agonistic antibody. These data demonstrate that ceramide links cellular stress responses induced by gamma-irradiation or anticancer drugs to the CD95 pathway of apoptosis.     

CD47 augments Fas/CD95-mediated apoptosis

Fas (CD95) mediates apoptosis of many cell types, but the susceptibility of cells to killing by Fas ligand and anti-Fas antibodies is highly variable. Jurkat T cells lacking CD47 (integrin-associated protein) are relatively resistant to Fas-mediated death but are efficiently killed by Fas ligand or anti-Fas IgM (CH11) upon expression of CD47. Lack of CD47 impairs events downstream of Fas activation including caspase activation, poly-(ADP-ribose) polymerase cleavage, cytochrome c release from mitochondria, loss of mitochondrial membrane potential, and DNA cleavage. Neither CD47 signaling nor raft association of CD47 is required to enable Fas apoptosis. CH11 induces association of Fas and CD47. Primary T cells from CD47-null mice are also protected from Fas-mediated killing relative to wild type T cells. Thus CD47 associates with Fas upon its activation and augments Fas-mediated apoptosis.     

Dendritic cells are resistant to apoptosis through the Fas (CD95/APO-1) pathway.

Immunoregulation of lymphocytes and macrophages in the peripheral immune system is achieved in part by activation-induced cell death. Members of the TNF receptor family including Fas (CD95) are involved in the regulation of activation-induced cell death. To determine whether activation-induced cell death plays a role in regulation of dendritic cells (DCs), we examined interactions between Ag-presenting murine DCs and Ag-specific Th1 CD4+ T cells. Whereas mature bone marrow- or spleen-derived DCs expressed high levels of Fas, these DCs were relatively insensitive to Fas-mediated killing by the agonist mAb, Jo-2, as well as authentic Fas ligand expressed on the CD4+ T cell line, A.E7. The insensitivity to Fas-mediated apoptosis was not affected by priming with IFN-gamma and/or TNF-alpha or by blocking the DC survival signals TNF-related activation-induced cytokine and CD40L. However, apoptosis could be induced with C2-ceramide, suggesting that signals proximal to the generation of ceramide might mediate resistance to Fas. Analysis of protein expression of several anti-apoptotic mediators revealed that expression of the intracellular inhibitor of apoptosis Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein was significantly higher in Fas-resistant DCs than in Fas-sensitive macrophages, suggesting a possible role for Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein in DC resistance to Fas-mediated apoptosis. Our results demonstrate that murine DCs differ significantly from other APC populations in susceptibility to Fas-mediated apoptosis during cognate presentation of Ag. Because DCs are most notable for initiation of an immune response, resistance to apoptosis may contribute to this function.     

Responsiveness of mouse corpora luteal cells to Fas antigen (CD95)-mediated apoptosis

Regression of the corpus luteum (CL) occurs by apoptosis. The Fas antigen (Fas) is a cell surface receptor that induces apoptosis in sensitive cells when bound to Fas ligand or agonistic anti-Fas monoclonal antibodies (Fas mAb). A potential role for Fas to induce apoptosis in dispersed CL cell preparations was tested in cells isolated from mice on Days 2-4 of pseudopregnancy. Total CL dispersates, containing steroidogenic luteal cells, fibroblasts, and endothelial cells, were cultured. The effect of pretreatment of cultures with cytokines interferon gamma (IFN) and tumor necrosis factor alpha (TNF) was examined because these cytokines demonstrated effects on Fas-mediated apoptosis in other cell types. Fas mAb had no effect on viability of CL cells cultured in 5% fetal bovine serum (FBS) and pretreated with or without IFN or TNF, but Fas mAb did kill 23% of the cells in cultures pretreated with IFN + TNF. Fas mRNA was detectable in cultured CL cells and was increased 2.1-, 2. 0-, and 11.8-fold by treatment with TNF, IFN, or IFN + TNF, respectively. CL cells treated with the protein synthesis inhibitor cycloheximide (CX) were killed by Fas mAb in the absence of cytokine pretreatment (34%); pretreatment with IFN or IFN + TNF further potentiated killing (62% and 96%, respectively), whereas pretreatment with TNF had no effect (42%). Cells cultured in medium supplemented with insulin, transferrin, and selenium instead of FBS were killed by Fas mAb in the presence of IFN (23%) or IFN + TNF (29%) but not in the presence of TNF. Cells derived from the mouse CL have a functional Fas pathway that is inhibited by FBS and activated by treatment with CX, IFN, and IFN + TNF.     

CD95/Fas mediates cognitive improvement after traumatic brain injury

CD95 （APO- 1/Fas）/CD95L （APO- 1L/FasL/CD 178） is a receptor/cytokine pair of the tumor necrosis factor/nerve growth factor （TNF/NGF） superfamily. Similar to other receptors of this family, activation of CD95/Fas results in the formation of a death-inducing signaling complex （DISC） and subsequent activation of the apoptotic cascade .[第一段]     

Engagement of CD4 before TCR triggering regulates both Bax- and Fas (CD95)-mediated apoptosis

In the present study, we have aimed at clarifying the CD4-dependent molecular mechanisms that regulate human memory T cell susceptibility to both Fas (CD95)-dependent and Bcl-2-dependent apoptotic pathways following antigenic challenge. To address this issue, we used an experimental system of viral and alloantigen-specific T cell lines and clones and two ligands of CD4 molecules, Leu-3a mAb and HIV gp120. We demonstrate that CD4 engagement before TCR triggering suppresses the TCR-mediated neosynthesis of the Flice-like inhibitory protein and transforms memory T cells from a CD95-resistant to a CD95-susceptible phenotype. Moreover, evidence that the apoptotic programs were executed while Fas ligand mRNA expression was inhibited led us to analyze Bcl-2-dependent pathways. The data show that the engagement of CD4 separately from TCR influences the expression of the proapoptotic protein Bax independently of the anti-apoptotic protein Bcl-2, whereas Ag activation coordinately modulates both Bax and Bcl-2. The increased expression of Bax and the consequent dissipation of the mitochondrial transmembrane potential (DeltaPsim) suggest a novel immunoregulatory function of CD4 and demonstrate that both passive cell death and activation-induced cell death are operative in CD4+ memory T cells. Furthermore, analysis of the mechanisms by which IL-2 and IL-4 cytokines exert their protective function on CD4+ T cells in the presence of soluble CD4 ligands shows that they were able to revert susceptibility to Bax-mediated but not to CD95-dependent apoptotic pathways.     

Manganese superoxide dismutase inactivation during Fas (CD95)-mediated apoptosis in Jurkat T cells

Manganese superoxide dismutase (MnSOD, SOD2) is an essential primary antioxidant enzyme which converts superoxide radical to hydrogen peroxide within the mitochondrial matrix. MnSOD plays a prominent role in protection against many apoptotic stimuli. Its absence may therefore impair the cellular redox balance and enhance apoptosis. Our data show that in Jurkat T cells, following oligomerization of the Fas receptor, MnSOD is selectively degraded during apoptosis. In the presence of cycloheximide, an inhibitor of protein synthesis, the rates of cell death and MnSOD degradation were accelerated. Fas-induced MnSOD cleavage was partially inhibited in the presence of the pan-caspase inhibitor, z-VAD-fmk. MnSOD in the mitochondrial fractions was cleaved in vitro by treatment with the cytosolic fraction of Fas-activated cells. Moreover, two possible cleavage sites of recombinant hMnSOD by direct interaction with recombinant caspase-3 were noted. Cellular and mitochondrial factors were found to be necessary for the interaction. These factors include intracellular mobilization of calcium. Our data indicate that inactivation of MnSOD in receptor-mediated apoptosis by caspase-specific degradation would render the mitochondria sensitive to the steady-state production of superoxide, decrease the steady-state flux of H₂O₂, expedite the loss of mitochondrial function, and potentiate apoptosis.     

Interferon-beta-induced activation of c-Jun NH2-terminal kinase mediates apoptosis through up-regulation of CD95 in CH31 B lymphoma cells

Type I interferon (IFN)-induced antitumor action is due in part to apoptosis, but the molecular mechanisms underlying IFN-induced apoptosis remain largely unresolved. In the present study, we demonstrate that IFN-beta induced apoptosis and the loss of mitochondrial membrane potential (delta psi m) in the murine CH31 B lymphoma cell line, and this was accompanied by the up-regulation of CD95, but not CD95-ligand (CD95-L), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL). Pretreatment with anti-CD95-L mAb partially prevented the IFN-beta-induced loss of delta psi m, suggesting that the interaction of IFN-beta-up-regulated CD95 with CD95-L plays a crucial role in the induction of fratricide. IFN-beta induced a sustained activation of c-Jun NH2-terminal kinase 1 (JNK1), but not extracellular signal-regulated kinases (ERKs). The IFN-beta-induced apoptosis and loss of delta psi m were substantially compromised in cells overexpressing a dominant-negative form of JNK1 (dnJNK1), and it was slightly enhanced in cells carrying a constitutively active JNK construct, MKK7-JNK1 fusion protein. The IFN-beta-induced up-regulation of CD95 together with caspase-8 activation was also abrogated in the dnJNK1 cells while it was further enhanced in the MKK7-JNK1 cells. The levels of cellular FLIP (c-FLIP), competitively interacting with caspase-8, were down-regulated by stimulation with IFN-beta but were reversed by the proteasome inhibitor lactacystin. Collectively, the IFN-beta-induced sustained activation of JNK mediates apoptosis, at least in part, through up-regulation of CD95 protein in combination with down-regulation of c-FLIP protein.     

A novel adenoviral vector expressing human Fas/CD95/APO-1 enhances p53-mediated apoptosis.

Recent evidence suggests an intriguing link between p53 and the Fas pathway. To evaluate this association further, we utilized a recombinant adenoviral vector (AdWTp53) to overexpress wild-type p53 in lung cancer (A549, H23, EKVX and HOP92) and breast cancer (MDA-MB-231 and MCF-7) cell lines and observed an increase in the Fas/CD95/APO-1 protein levels. Furthermore, this increase correlated with the sensitivity of the cell lines to p53-mediated cytotoxicity. To examine the effects of Fas over-expression in cells resistant to p53 over-expression, we constructed AdFas, an adenoviral vector capable of transferring functional human Fas to cancer cells. Interestingly, infection of p53-resistant MCF-7 cells with AdFas sensitized them to p53-mediated apoptosis. These studies indicate that combined over-expression of Fas and wild-type p53 may be an effective cancer gene therapy approach, especially in cells relatively resistant to p53 over-expression.     

Fas/CD95 is associated with glucocorticoid-induced osteocyte apoptosis

Prolonged use of glucocorticoids is associated with decreased bone formation, increased resorption and osteonecrosis, through direct and indirect effects on the activity and viability of bone effector cells, osteoblasts and osteoclasts, and osteocytes. This study has investigated molecular pathways implicated in Dexamethasone-induced apoptosis of osteocytes, using a cell line and primary chicken cells. MLO-Y4 osteocytes were pre-treated with several bisphosphonates representing a range of anti-resorptive activities and conformation/structure relationships, and were subsequently challenged with Dexamethasone. Apoptotic cells were detected at various times after treatment using morphological and biochemical criteria. Dex was shown to induce apoptosis associated with the Fas/CD95 death receptor and in a caspase 8 dependent manner. The apoptotic response was inhibited by all variants of the BP molecules, including those with reduced anti-resorptive activity, indicating that Dex-induced apoptosis is independent of anti-osteoclastic activity. Dex-induced apoptosis was associated with a transient increase in phosphorylated ERK 1/2 and was blocked by the ERK inhibitor UO126. In addition, both UO126 and BPs decreased localization of Fas to the cell membrane. ERK activation by PMA did not induce death or Fas upregulation, suggesting that Fas may be important for the induction of apoptosis and the existence of an additional factor activated by Dex which enables the cooperation between the Dex-activated ERK and Fas pathways, during apoptosis of osteocytes. Furthermore, upregulation of death and Fas was not accompanied by upregulation of FasL, pointing to the possible existence of FasL-independent Fas-associated death in these cells.     

The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems

Heat shock protein 70 (hsp70) is a stress-inducible protein that prevents apoptosis induced by a wide range of cytotoxic agents by an as yet undefined mechanism. The caspase family of cysteine proteases have been attributed a central role in the execution of apoptosis. However, several cases of caspase-independent apoptosis have been recently reported, suggesting that caspases may not be necessary for apoptosis in all cells. This study examines the protective role of hsp70 in both caspase-dependent and -independent apoptosis. Hydrogen peroxide (H2O2) used at low and high concentrations in Jurkat T cells induces caspase-dependent and -independent apoptosis, respectively. A hsp70-transfected Jurkat clone was used to observe the protection mediated by hsp70 during these two forms of apoptosis. Results reveal that hsp70 inhibits both caspase-dependent and -independent apoptosis. Furthermore, measurement of caspase-3 activity during caspase-dependent apoptosis revealed that caspase activation was inhibited in hsp70 transfectants. Early apoptotic events, such as mitochondrial depolarization, cytochrome c release, and increased intracellular calcium, were demonstrated to be common to both caspase-dependent and -independent H2O2-induced apoptosis. The inhibition of these events by hsp70 suggests that hsp70 may be an important anti-apoptotic regulator, functioning at a very early stage in the apoptotic pathway.     

Carbon monoxide promotes Fas/CD95-induced apoptosis in Jurkat cells

A properly functioning immune system is dependent on programmed cell death/apoptosis at virtually every stage of lymphocyte development and activity. Carbon monoxide (CO), an enzymatic product of heme oxyenase-1, has been shown to possess anti-apoptotic effects in a number of different model systems. The purpose of the present study was to expand on this knowledge to determine the role of CO in the well established model of Fas/CD95-induced apoptosis in Jurkat cells, and to determine the mechanism by which CO can modulate T-cell apoptosis. Exposure of Jurkat cells to CO resulted in augmentation in Fas/CD95-induced apoptosis, which correlated with CO-induced up-regulation of the pro-apoptotic protein FADD as well as activation of caspase-8, -9, and -3 while simultaneously down-regulating the anti-apoptotic protein BCL-2. These effects of CO were lost with overexpression of the small interfering RNA of FADD. CO, as demonstrated previously in endothelial cells, was also anti-apoptotic in Jurkat cells against tumor necrosis factor and etoposide. We further demonstrate that this pro-apoptotic effect of CO was independent of reactive oxygen species production and involved inhibition in Fas/CD95-induced activation of the pro-survival ERK MAPK. We conclude that in contrast to other studies showing the anti-apoptotic effects of CO, Fas/CD95-induced cell death in Jurkat cells is augmented by exposure to CO and that this occurs in part via inhibition in the activation of ERK MAPK. These data begin to elucidate specific differences with regard to the effects of CO and cell death pathways and provide important and valuable insight into potential mechanisms of action.     

CD95(Fas/APO-1) signals ceramide generation independent of the effector stage of apoptosis

Although numerous studies document caspase-independent ceramide generation preceding apoptosis upon environmental stress, the molecular ordering of ceramide generation during cytokine-induced apoptosis remains uncertain. Here, we show that CD95-induced ceramide elevation occurs during the initiation phase of apoptosis. We titrated down the amount of FADD transfected into HeLa and 293T cells until it was insufficient for apoptosis, although cycloheximide (CHX) still triggered the effector phase. Even in the absence of CHX, ceramide levels increased rapidly, peaking at 2.7 +/- 0.2-fold of control 8 h post-transfection. Dominant negative FADD failed to confer ceramide generation or CHX-mediated apoptosis. Ceramide generation induced by FADD was initiator caspase-dependent, being blocked by crmA. Limited pro-caspase 8 overexpression also increased ceramide levels 2.7 +/- 0.2-fold, yet failed, without CHX, to initiate apoptosis. Expression of membrane-targeted oligomerized CD-8 caspase 8 induced apoptosis without CHX, yet elevated ceramide only to a level equivalent to limited pro-caspase 8 transfection. Ceramide elevations were detected concurrently by diacylglycerol kinase and electrospray tandem mass spectrometry. These investigations provide evidence that ceramide generation is initiator caspase-dependent and occurs prior to commitment to the effector phase of apoptosis, definitively ordering ceramide as proximal in CD95 signaling.     

Differential effect of IL-18 on endothelial cell apoptosis mediated by TNF-alpha and Fas (CD95)

Interleukin-18 (IL-18) is a newly identified cytokine with proinflammatory activity. Numerous studies have shown that proinflammatory cytokines may regulate endothelial cells (EC) apoptosis mediated by members of the tumor necrosis factor (TNF) family, such as TNF-alpha and Fas. In this study we hypothesized that IL-18 may regulate the susceptibility of liver endothelial cells (LEC) to apoptosis induced by TNF and Fas. IL-18 increased the susceptibility of LEC to undergo apoptosis mediated by TNF but not by Fas. Since TNF-induced apoptosis is mediated by the type I TNF receptor (TNFRI), we investigated up-regulation of this receptor in IL-18-treated LEC. IL-18 induced up-regulation of the TNFRI on the surface of LEC. Partial blocking of LEC apoptosis induced by IL-18 and TNF was observed when the cells were pretreated with the broad-spectrum inhibitor of caspases z-VAD-fmk, suggesting involvement of the caspase pathway in apoptosis induced by these cytokines in these cells. Our results show that IL-18 differentially regulates apoptosis mediated by the death-inducing factors, TNF and Fas. To our knowledge, this is the first report that IL-18 may regulate endothelial cell apoptosis mediated by TNF. These results may have clinical implications in those clinical hepatic conditions associated with high levels of IL-18 and TNF.     

TCR engagement regulates differential responsiveness of human memory T cells to Fas (CD95)-mediated apoptosis

In this work, we have tried to establish whether human memory T cells may be protected from Fas (CD95)-induced apoptosis when correctly activated by Ag, and not protected when nonspecifically or incorrectly activated. In particular, we wanted to investigate the molecular mechanisms that regulate the fate of memory T cells following an antigenic challenge. To address this issue, we chose an experimental system that closely mimics physiological T cell activation such as human T cell lines and clones specific for viral peptides or alloantigens. We demonstrate that memory T cells acquire an activation-induced cell death (AICD)-resistant phenotype when TCRs are properly engaged by specific Ag bound to MHC molecules. Ag concentration and costimulation are critical parameters in regulating the protective effect. The analysis of the mechanisms involved in the block of CD95 signal transduction pathways revealed that the crucial events are the inhibition of CD95-associated IL-1beta-converting enzyme (ICE)-like protease (FLICE) activation and poly(ADP)-ribose polymerase cleavage, and the mRNA expression of FLICE-like inhibitory protein. Furthermore, we have observed that TCR-mediated neosynthesis of FLICE-like inhibitory protein mRNA is suppressed either by protein tyrosine kinase inhibitors or cyclosporin A. In conclusion, the present analysis of the effects of TCR triggering on the regulation of AICD suggests that AICD could be inhibited in human memory T cells activated in vivo by a foreign Ag, but may become operative when the Ag has been cleared.     

Modification of tumor cells with Fas (CD95) antigen gene and Fas ligand (CD95L) gene transfection by electroporation for immunotherapy of cancer

Electroporation is a method for introducing DNA into cells by using a high-voltage electric field. This method is very simple and easily manipulated. We describe here a method for the modification of tumor cells with the Fas/Apo-1 (CD95) antigen-gene and Fas ligand (FasL)-gene transfection through the use of electroporation, and suggest that the Fas-FasL system is a good target for the induction of apoptosis-mediated antitumor activity. The Fas receptor/ligand system induces apoptosis and plays an important role in regulation of the immune system. In the method described, hepatoma MH134 (Fas⁻ and FasL⁻) is transfected with murine Fas and FasL cDNA. A single administration of monoclonal anti-Fas antibody efficiently suppresses the growth of F6b (MH134+Neo+Fas) tumors but not that of N1d (MH134+Neo) tumors in gld/gld lpr/lpr mice. MH134+Neo+FasL tumor cells were rejected after the induction of inflammation with infiltration of neutrophils in mice. These results suggest that electroporation and Fas-mediated apoptosis are a good method for inducing of antitumor activity.     

Nitric oxide negatively regulates Fas CD95-induced apoptosis through inhibition of ubiquitin-proteasome-mediated degradation of FLICE inhibitory protein

Stimulation of cell surface Fas (CD95) results in recruitment of cytoplasmic proteins and activation of caspase-8, which in turn activates downstream effector caspases leading to programmed cell death. Nitric oxide (NO) plays a key role in the regulation of apoptosis, but its role in Fas-induced cell death and the underlying mechanism are largely unknown. Here we show that stimulation of the Fas receptor by its ligand (FasL) results in rapid generation of NO and concomitant decrease in cellular FLICE inhibitory protein (FLIP) expression without significant effect on Fas and Fas-associated death domain (FADD) adapter protein levels. FLIP down-regulation as well as caspase-8 activation and apoptosis induced by FasL were all inhibited by the NO-liberating agent sodium nitroprusside and dipropylenetriamine NONOate, whereas the NO synthase inhibitor aminoguanidine and NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) had opposite effects, indicating an anti-apoptotic role of NO in the Fas signaling process. FasL-induced down-regulation of FLIP is mediated by a ubiquitin-proteasome pathway that is negatively regulated by NO. S-nitrosylation of FLIP is an important mechanism rendering FLIP resistant to ubiquitination and proteasomal degradation by FasL. Deletion analysis shows that the caspase-like domain of FLIP is a key target for S-nitrosylation by NO, and mutations of its cysteine 254 and cysteine 259 residues completely inhibit S-nitrosylation, leading to increased ubiquitination and proteasomal degradation of FLIP. These findings indicate a novel pathway for NO regulation of FLIP that provides a key mechanism for apoptosis regulation and a potential new target for intervention in death receptor-associated diseases.