Apoptosis in human embryo development: 1. Cerebral cortex

We investigated the apoptosis at the beginning of human cerebral cortex development, in the 6th week of embryogenesis, Carnegie stages 16 and 17. Attention was focused on the dorsal wall of the telencephalon to the ventricular zone of proliferation and to the postmitotic zone with beginning of neuronal migration. We identified apoptotic cells in tissue sections by propidium iodide staining, TUNEL and immunohistochemistry for Fas(APO-1/CD95). We determined the distribution and the percentage (reported to the propidium iodide stained nuclei) of apoptotic TUNEL-positive and Fas(APO-1/CD95)-positive cells. TUNEL-positive apoptotic cells in the proliferative zone were 20% in stage 16 and 60% in stage 17. TUNEL-positive apoptotic cells in the postmitotic zone were 8% in stage 16 and 30% in stage 17. CD95-positive apoptotic cells in the proliferative zone were 5% in stage 16 and 2% in stage 17. There were no CD95-positive cells in the postmitotic zone. We evidentiated the presence of the suicide receptor Fas(APO-1/CD95) only on a small population of apoptotic neuroblasts in the proliferative zone. The differences between apoptotic distribution and receptors in early corticogenesis suggest that different apoptotic pathways drive the selection of neuronal populations.     

Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.

Activation of the cell surface receptor Fas/APO-1 (CD95) induces apoptosis in lymphocytes and regulates immune responses. The cytoplasmic membrane protein Bcl-2 inhibits lymphocyte killing by diverse cytotoxic agents, but we found it provided little protection against Fas/APO-1-transduced apoptosis in B lymphoid cell lines, thymocytes and activated T cells. In contrast, the cowpox virus protease inhibitor CrmA blocked Fas/APO-1-transduced apoptosis, but did not affect cell death induced by gamma-radiation or serum deprivation. Signalling through Fas/APO-1 did not down-regulate Bcl-2 or induce its antagonists Bax and Bcl-xS. In Fas/APO-1-deficient lpr mice, Bcl-2 transgenes markedly augmented the survival of antigen-activated T cells and the abnormal accumulation of lymphocytes (although they did not interfere with deletion of auto-reactive cells in the thymus). These data raise the possibility that Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.     

A critical role of glutathione in determining apoptosis sensitivity and resistance in leukemia cells

In chemosensitive leukemias and solid tumors, anticancer drugs have been shown to induce apoptosis. Deficiencies in the apoptotic pathways may lead to chemoresistance. Here we report that glutathione (GSH) plays a critical role in activation of apoptosis pathways by CD95 (APO-1/Fas) or anticancer drugs. Upon treatment with anticancer drugs or CD95 triggering, CD95-resistant or Bcl-x(L) overexpressing CEM cells were deficient in activation of apoptosis pathways. CD95-resistant and Bcl-x(L) overexpressing CEM cells exhibited higher intracellular GSH levels in comparison to parental cells. Downregulation of GSH by L-buthionine-(S,R)-sulfoxime (BSO), a specific inhibitor of glutathione synthesis, reversed deficiencies in activation of apoptosis pathways by anticancer drugs or CD95. Interestingly, downregulation of GSH by BSO increased CD95 DISC formation in type I cells. In hybrids of CD95-resistant cells with sensitive cells and hybrids of overexpressing Bcl-x(L) cells with sensitive cells, the phenotype of apoptosis resistance was dominant. Also, in these hybrids, downregulation of GSH reversed CD95- and chemoresistance. We conclude that dominant apoptosis resistance depends, at least in part, on intracellular GSH levels, which may affect apoptosis signaling at different compartments, for example, the death receptor or mitochondria.     

Regulation of death receptor-induced apoptosis induced via CD95/Fas and other death receptors

Apoptosis (programmed cell death) is common to all multicellular organisms. Apoptosis plays a central role in cell differentiation, removal of damaged cells, and the homeostasis of the immune system. There are two apoptosis signal pathways: the extrinsic (transmitted through death receptors (DR)) or the intrinsic (mitochondrial) death pathways. A death receptor, CD95 (Fas/APO-1), was discovered 20 years ago. This review is focused on the mechanisms of death receptor-induced apoptosis via CD95 (Fas/APO-1)-mediated apoptosis and the role of the antiapoptotic protein c-FLIP in the extrinsic apoptosis regulation. The regulation of this pathway is crucial for the immune system. Defects in the regulation of CD95-mediated result in serious diseases such as cancer, autoimmunity, and AIDS. Therefore, gaining insights into apoptosis will have wide implications for developing approaches to treatment strategies of these diseases.     

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.     

Apoptosis in the immune system: 1. Fas-induced apoptosis in monocytes-derived human dendritic cells

Dendritic cells (DC) are cells of the hematopoietic system specialized in capturing antigens and initiating T cell-mediated immune responses. We show here that human DC generated from adherent peripheral blood mononuclear cells (PBMC) after in vitro stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) express Fas antigen (APO-1, CD95) and can undergo apoptosis upon triggering of Fas by monoclonal antibodies. Immature monocytes-derived dendritic cells (MDDC) upregulate CD86 and HLA-DR expression and develop dendrites and veiled processes. Flow cytometry analysis revealed CD95 expression in approx. 40% of these MDDC and incubation with anti-CD95 mAb (0.5μg/ml) induced apoptosis when compared to untreated controls. The extent of apoptosis induced by the agonist anti-Fas antibody strongly related to the percentage of cells expressing CD 95. Upon tumor necrosis factor α (TNF-α) additional stimulation, MDDC assumed a characteristic mature dendritic cells morphology showing prolonged veils, CD83 expression, and high levels of HLA-DR. These cells have downregulated their Fas receptors (to approx. 20%) and undergo apoptosis to a lesser extent when treated with anti-CD 95, as demonstrated by the hardly noticeable effect of this antibody on the viability of cultured cells as compared to controls. Thus, upon TNF-α induced maturation, MDDC became resistant to Fas-induced apoptosis. The apoptotic episodes surrounding the earlier stage of DC differentiation appeared to be mediated by Fas. In contrast, a Fas independent pathway is probably responsible for the apoptotic events associated with terminally differentiated DC.     

Regulation of CD95/Fas signaling at the DISC

CD95 (APO-1/Fas) is a member of the death receptor (DR) family. Stimulation of CD95 leads to induction of apoptotic and non-apoptotic signaling pathways. The formation of the CD95 death-inducing signaling complex (DISC) is the initial step of CD95 signaling. Activation of procaspase-8 at the DISC leads to the induction of DR-mediated apoptosis. The activation of procaspase-8 is blocked by cellular FLICE-inhibitory proteins (c-FLIP). This review is focused on the role in the CD95-mediated signaling of the death effector domain-containing proteins procaspase-8 and c-FLIP. We discuss how dynamic cross-talk between procaspase-8 and c-FLIP at the DISC regulates life/death decisions at CD95.     

Fas-induced apoptosis in B cells

Engagement of the cell surface receptor Fas/APO-1 (CD95) initiates a sequence of intracellular events that leads to apoptotic cell death, and this outcome occurs in B cells as it does in other cell types. Fas signaling for B cell death is of particular interest because the expression and function of Fas is altered by engagement of additional cell surface receptors, leading to marked receptor-specific variation in susceptibility to Fas-induced apoptosis. Evidence suggests that the sensitivity of B cells to Fas-mediated apoptosis is intimately connected to homeostasis in the serological arm of the immune system and plays a role in the dysregulation that occurs in certain autoimmune and malignant dyscrasias.     

The relevance of NF-?B for CD95 Signaling in Tumor Cells

Most members of the death receptor family including CD95 (APO-1/Fas) have beenshown to induce both apoptosis as well as nonapoptotic pathways depending on thetissue and the circumstances. One of the nonapoptotic pathways emanating from CD95,activation of NF-?B, has recently been demonstrated to regulate invasiveness ofapoptosis resistant tumor cells. In contrast, activation of NF-?B in apoptosing cells isbelieved to be suppressed due to cleavage of various NF-?B pathway components byactive caspases that execute apoptosis. We now present data demonstrating that incertain highly CD95 apoptosis sensitive cells NF-?B is robustly activated. In factoverexpression of apoptosis inhibitors such as Bcl-2 or c-FLIPL in these cells results indecreased activation of NF-?B through CD95. We propose a model in which NF-?B isgenerally activated in certain cells but may have different functions depending onwhether cells are programmed to die or to survive.     

Apoptosis in human embryo development: 3. Fas-induced apoptosis in brain primary cultures

Fas (APO-1/CD95) is an important apoptotic mediator for both immune and nervous systems. In the present study, we have investigated the expression and function of Fas in human embryonic/fetal brain primary cultures from 12 human embryos and fetuses with gestational ages between 5 to 22 weeks. Anti-Fas fluorescent antibody was used for labeling of Fas positive cells and for quantitation of Fas expression in brain cultures. To demonstrate that Fas receptor is functional in human embryonic/fetal brain cells, anti-Human-Fas monoclonal antibody (0.5 μg/ml) was used to induce apoptosis in brain primary cultures. Apoptosis was investigated by flow-cytometry and fluorescent microscopy using TUNEL and annexin V labeling. Fas was found to be expressed in the embryonic/fetal human primary brain cultures, on neuronal and glial cells or their precursors, varying with gestational ages. Cross-linking of Fas induced apoptosis in brain cultures indicating that Fas receptor functions as a death receptor. We also showed that cell death triggered through Fas receptor was caspase dependent, hence it was blocked by a selective caspase-8 inhibitor (IETD-fmk).These results suggest that Fas is involved in neuronal apoptosis in the developing human brain.     

Modulation of the CD95-induced apoptosis: the role of CD95 N-glycosylation

Protein modifications of death receptor pathways play a central role in the regulation of apoptosis. It has been demonstrated that O-glycosylation of TRAIL-receptor (R) is essential for sensitivity and resistance towards TRAIL-mediated apoptosis. In this study we ask whether and how glycosylation of CD95 (Fas/APO-1), another death receptor, influences DISC formation and procaspase-8 activation at the CD95 DISC and thereby the onset of apoptosis. We concentrated on N-glycostructure since O-glycosylation of CD95 was not found. We applied different approaches to analyze the role of CD95 N-glycosylation on the signal transduction: in silico modeling of CD95 DISC, generation of CD95 glycosylation mutants (at N136 and N118), modulation of N-glycosylation by deoxymannojirimycin (DMM) and sialidase from Vibrio cholerae (VCN). We demonstrate that N-deglycosylation of CD95 does not block DISC formation and results only in the reduction of the procaspase-8 activation at the DISC. These findings are important for the better understanding of CD95 apoptosis regulation and reveal differences between apoptotic signaling pathways of the TRAIL and CD95 systems.     

Apoptogenic ganglioside GD3 directly induces the mitochondrial permeability transition.

Early events in apoptotic cascades initiated by ceramides or by activation of the surface receptor CD95 (Fas/APO-1) include the formation of ganglioside GD3. GD3 appears to be both necessary and sufficient to propagate this lipid-mediated apoptotic pathway. Later events common to many apoptotic pathways include induction of the mitochondrial permeability transition (PT) and cytochrome c release, which in turn triggers downstream caspases and cell death. The links between GD3 formation and downstream stages of apoptosis are unknown. We report that ganglioside GD3 directly induces the PT in isolated rat liver mitochondria at 30-100 microM in the presence of exogenous substrate (succinate) and at approximately 3 microM in the absence of exogenous substrate. In contrast, other gangliosides tested (e.g. GM1) have only weak stimulatory effects in the presence of succinate and protect against PT induction in the absence of respiratory substrates. GD3-mediated induction of PT was antagonized by known PT inhibitors, namely cyclosporin A, ADP, trifluoperazine, and Mg(2+). GD3 induced PT even in the presence of submicromolar Ca(2+); GD3 is therefore the first biological PT inducer identified that does not require elevated Ca(2+). Exposure to GD3 also led to mitochondrial cytochrome c release. In contrast, C(2)-ceramide, which can initiate the lipid-mediated apoptotic cascade in susceptible cells, failed to either induce PT or release cytochrome c. These observations suggest that GD3 propagates apoptosis by inducing the PT and cytochrome c release. This model provides a mechanistic link between the earlier and later stages of CD95-induced/ceramide-mediated apoptosis.     

Recruitment of apoptotic cysteine proteases (caspases) in influenza virus-induced cell death.

Influenza virus infection induces apoptosis in cultured cells with an augmented expression of Fas (APO-1/CD95). Caspases, a family of cysteine proteases structurally related to interleukin-1-beta-converting enzyme (ICE), play crucial roles in apoptosis induced by various stimuli, including Fas. However, activation of the caspase-cascade seems to be different in various pathways of apoptotic stimuli. We therefore examined the involvement of caspases in influenza virus-induced apoptosis using caspase inhibitors. We found that z-VAD-fmk and z-IETD-fmk effectively inhibited virus-induced apoptosis, whereas Ac-DEVD-CHO and Ac-YVAD-CHO showed partial and little effect on virus-induced cell death, respectively. Consistently, caspase-3-like activity, but not caspase-1-like activity, was increased in the virus-infected cells. The transfection of plasmids encoding viral inhibitors of caspase (v-FLIP or crmA) into HeLa cells inhibited apoptosis by virus infection. The peptide inhibitors of caspases used in this study did not inhibit viral replication. We conclude that influenza virus infection activates some caspases, and that this activation may be downstream of viral replication.     

Down-regulation of Fas-mediated apoptosis by plasma transglutaminase factor XIII that catalyzes fetal-specific cross-link of the Fas molecule

The Fas antigen, also designated as APO-1 or CD95, is a member of the tumor necrosis factor receptor superfamily and can mediate apoptotic cell death in various cells. We report here that blood coagulation factor XIII (plasma transglutaminase, fibrin stabilizing factor) inhibits apoptosis induced by a cytotoxic anti-Fas monoclonal antibody in Jurkat cells. When cells were treated with the antibody in fetal calf serum-containing media, higher-molecular-weight (180 K) polypeptides containing Fas molecule were detected by immunoblotting. Under conditions where the transglutaminase activity was eliminated or suppressed, the cross-link of Fas was not observed, and concurrently cell death was hastened. Moreover, an antibody against factor XIII strongly accelerated the Fas-mediated apoptosis. Furthermore, addition of partially purified factor XIII neutralized the apoptosis-promoting effect of anti-factor XIII antibody, indicating that this enzyme is involved in cross-link of Fas and down-regulates Fas-mediated apoptotic cell death. Significantly, the cross-link of Fas was seen only in fetal calf serum but not in newly-born calf serum, 1-year-old calf serum or adult bovine serum. These data suggest that plasma transglutaminase factor XIII may play a key role in fetal development of vertebrates via cross-link of Fas antigen.     

Common regulation of apoptosis signaling induced by CD95 and the DNA-damaging stimuli etoposide and gamma-radiation downstream from caspase-8 activation.

The death receptor CD95 (APO-1/Fas), the anticancer drug etoposide, and gamma-radiation induce apoptosis in the human T cell line Jurkat. Variant clones selected for resistance to CD95-induced apoptosis proved cross-resistant to etoposide- and radiation-induced apoptosis, suggesting that the apoptosis pathways induced by these distinct stimuli have critical component(s) in common. The pathways do not converge at the level of CD95 ligation or caspase-8 signaling. Whereas caspase-8 function was required for CD95-mediated cytochrome c release, effector caspase activation, and apoptosis, these responses were unaffected in etoposide-treated and irradiated cells when caspase-8 was inhibited by FLIPL. Both effector caspase processing and cytochrome c release were inhibited in the resistant variant cells as well as in Bcl-2 transfectants, suggesting that, in Jurkat cells, the apoptosis signaling pathways activated by CD95, etoposide, and gamma-radiation are under common mitochondrial control. All three stimuli induced ceramide production in wild-type cells, but not in resistant variant cells. Exogenous ceramide bypassed apoptosis resistance in the variant cells, but not in Bcl-2-transfected cells, suggesting that apoptosis signaling induced by CD95, etoposide, and gamma-radiation is subject to common regulation at a level different from that targeted by Bcl-2.     

Does CD95 have tumor promoting activities?

CD95 (APO-1/Fas) is an important inducer of the extrinsic apoptosis signaling pathway and therapy induced apoptosis of many tumor cells has been linked to the activity of CD95. Changes in the expression of CD95 and/or its ligand CD95L are frequently found in human cancer. The downregulation or mutation of CD95 has been proposed as a mechanism by which cancer cells avoid destruction by the immune system through reduced apoptosis sensitivity. CD95 has therefore been viewed as a tumor suppressor. Furthermore, increased CD95L concentration in tumor patients has been linked to tumor cells killing infiltrating lymphocytes in a process called "the tumor counter-attack". Recent data have illuminated unknown activities of CD95 in tumor cells with downregulated or mutated CD95 in the presence of increased CD95L. Under these conditions the stimulation of CD95 signals nonapoptotic pathways, activating NF-kappaB and MAP kinases for example, which may result in the induction of tumorigenic or prosurvival genes. A new model of CD95 functions is proposed in which CD95 is converted from a tumor suppressor to a tumor promotor by a single point mutation in one of the CD95 alleles, a situation frequently found in advanced human cancer, resulting in apoptosis resistance and activation of tumorigenic pathways.     

Fas activates NF-kappaB and induces apoptosis in T-cell lines by signaling pathways distinct from those induced by TNF-alpha

The p55 tumor necrosis factor (TNF) receptor and the Fas (CD95/APO-1) receptor share an intracellular domain necessary to induce apoptosis, suggesting they utilize common signaling pathways. To define pathways triggered by Fas and TNF-alpha we utilized human CEM-C7 T-cells. As expected, stimulation of either receptor induced apoptosis and TNF-alpha-induced signaling included the activation of NF-kappaB. Surprisingly, Fas-induced signaling also triggered the activation of NF-kappaB in T cells, yet the kinetics of NF-kappaB induction by Fas was markedly delayed. NF-kappaB activation by both pathways was persistent and due to the sequential degradation of IkappaB-alpha and IkappaB-beta. However, the kinetics of IkappaB degradation were different and there were differential effects of protease inhibitors and antioxidants on NF-kappaB activation. Signaling pathways leading to activation of apoptosis were similarly separable and were also independent of NF-kappaB activation. Thus, the Fas and TNF receptors utilize distinct signal transduction pathways in T-cells to induce NF-kappaB and apoptosis.