Activation of the CD95 (APO-1/Fas) pathway in drug- and gamma-irradiation-induced apoptosis of brain tumor cells

Chemotherapeutic agents and gamma-irradiation used in the treatment of brain tumors, the most common solid tumors of childhood, have been shown to act primarily by inducing apoptosis. Here, we report that activation of the CD95 pathway was involved in drug- and gamma-irradiation-induced apoptosis of medulloblastoma and glioblastoma cells. Upon treatment CD95 ligand (CD95-L) was induced that stimulated the CD95 pathway by crosslinking CD95 via an autocrine/paracrine loop. Blocking CD95-L/receptor interaction using F(ab')2 anti-CD95 antibody fragments strongly reduced apoptosis. Apoptosis depended on activation of caspases (interleukin 1beta-converting enzyme/Ced-3 like proteases) as it was almost completely abrograted by the broad range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Apoptosis was mediated by cleavage of the receptor proximal caspase FLICE/MACH (caspase-8) and the downstream caspase CPP32 (caspase-3, Apopain) resulting in cleavage of the prototype caspase substrate PARP. Moreover, CD95 was upregulated in wild-type p53 cells thereby increasing responsiveness towards CD95 triggering. Since activation of the CD95 system upon treatment was also found in primary medulloblastoma cells ex vivo, these findings may have implications to define chemosensitivity and to develop novel therapeutic strategies in the management of malignant brain tumors.     

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.     

Lung carcinomas do not induce T-cell apoptosis via the Fas/Fas ligand pathway but down-regulate CD3 epsilon expression

Background Non-small cell lung carcinoma (NSCLC) patients have impaired cellular immune responses. It has been hypothesized that tumor cells expressing Fas Ligand (FasL) induce in T lymphocytes: (a) apoptosis (tumor counterattack) and (b) down-regulation of CD3ζ expression. However, the hypothesis of tumor counterattack is still controversial. Methods We analyzed FasL expression on NSCLC cell lines and on tumor cells from lung adenocarcinoma patients by flow cytometry and immunocytochemistry. FasL mRNA expression was detected in NSCLC cell lines using RT-PCR, and functional FasL was evaluated on Fas-expressing Jurkat T-cells by annexin-V-FITC staining and by SubG₁ peak detection. Also, the proapoptotic effect of microvesicles released from NSCLC cell lines in Jurkat T-cells was studied. Alterations in the expression levels of CD3ζ, CD3ε, and CD28 [measured as mean fluorescence intensity (MFI)] were determined in Jurkat T-cells after co-culture with NSCLC cell lines or tumor-derived microvesicles. Furthermore, the expression levels of CD3ζ and CD3ε in CD4+T and CD8+T lymphocytes from lung adenocarcinoma patients was studied. Results Our results indicate that NSCLC cells neither FasL expressed nor induced apoptosis in Jurkat T-cells. Tumor-derived microvesicles did not induce apoptosis in Jurkat T-cells. In contrast, NSCLC cell lines down-regulated CD3ε but not CD3ζ chain expression in Jurkat T-cells; this effect was induced by soluble factors but not by microvesicles. In lung adenocarcinoma patients, significant decreases of MFI values for CD3ε, but not CD3ζ, were found in CD4+T and CD8+T cells from pleural effusion compared to peripheral blood and in peripheral blood of patients compared to healthy donors. Conclusions Our data do not support the tumor counterattack hypothesis for NSCLC. Nonetheless, down-regulation of CD3ε in T-cells induced by NSCLC cells might lead to T-cell dysfunction.     

Cutting edge: regulatory T cells do not mediate suppression via programmed cell death pathways

Regulatory T cells (Tregs) play a critical role in the immune system to regulate peripheral tolerance and prevent autoimmunity. However, the relative importance of different mechanisms of Treg function remains obscure. In this article, we reveal a limited role for programmed cell death pathways in mediating Treg suppression of conventional T cells. We show that Tregs are able to suppress the proliferation of conventional T cells that are resistant to apoptosis (Bim(-/-), Bim(-/-)Puma(-/-), Bcl-2 transgenic) or receptor-interacting serine-threonine kinase-dependent necrosis (also referred to as regulated necrosis or necroptosis) (Ripk3(-/-)) in several in vitro and in vivo assays. These data suggest that programmed cell death pathways, such as apoptosis and receptor-interacting serine-threonine kinase-dependent necrosis, are not required for Treg-mediated suppression.     

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.     

Asymmetric dimethylarginine attenuates serum starvation-induced apoptosis via suppression of the Fas (APO-1/CD95)/JNK (SAPK) pathway

Asymmetric dimethylarginine (ADMA) is synthesized by protein arginine methyltransferases during methylation of protein arginine residues and released into blood upon proteolysis. Higher concentrations of ADMA in blood have been observed in patients with metabolic diseases and certain cancers. However, the role of ADMA in colon cancer has not been well investigated. ADMA serum levels in human patients diagnosed with colon cancer were found to be higher than those present in healthy subjects. ADMA treatment of LoVo cells, a human colon adenocarcinoma cell line, attenuated serum starvation-induced apoptosis and suppressed the activation of the Fas (APO-1/CD95)/JNK (SAPK) (c-Jun N terminal protein kinase/stress-activated protein kinase)pathway. ADMA also suppressed the activation of JNK triggered by death receptor ligand anti-Fas mAb and exogenous C₂-ceramide. Moreover, we demonstrated that ADMA pretreatment protected LoVo cells from doxorubicin hydrochloride-induced cell death and activation of the Fas/JNK pathway. In summary, our results suggest that the elevated ADMA in colon cancer patients may contribute to the blocking of apoptosis of cancer cells in response to stress and chemotherapy.     

Looking beneath the surface: the cell death pathway of Fas/APO-1 (CD95).

SUMMARY: The biochemical basis of programmed cell death is poorly understood in mammals. The cell surface receptor Fas/APO-1 (CD95) is one molecule known to be central to a number of mammalian cell death processes. Several studies in the past year have led to insights about the role of Fas/APO-1 in vivo and have also given some clues about the biochemical components of the Fas/APO-1 death pathway. This article reviews those studies and discuss models of Fas/APO-1 signaling and function. BACKGROUND: Cell death occurs as a normal process in a wide variety of developmental and homeostatic contexts in metazoan organisms (1); it represents the timely and appropriate fate for many or even the majority of cells born in certain organ systems. Despite the importance and ubiquitous nature of such physiologic, or "programmed", cell death, little is known about the molecular events that mediate this process. That a conserved biochemical pathway exists is suggested by the observation that programmed cell death is almost always accompanied by a consistent set of morphologic changes, an appearance known as apoptosis (2). The identification of the genes that control programmed cell death in higher eukaryotes has been hampered by several inherent difficulties. First, the genetic tools so useful in dissecting cell death pathways in Caenorhabditis elegans (3) and Drosophila (4) have not been available in higher eukaryotes. Second, the death-inducing properties of such genes makes genetic selection an impractical means of identification. Third, it appears that many cell death genes are constitutively expressed and present in an inactive form (5), making it unlikely that they could be discovered by techniques relying upon differential gene expression. Finally, genes identified by virtue of an ability to induce death when overexpressed must be subjected to rigorous criteria to determine whether the cell death is of physiologic importance, since it is likely that overexpression of certain proteins may lead to toxic effects that are distinct from the in vivo roles of those proteins. Two approaches to date have yielded the most information about cell death processes: (i) identification of cell death genes by classical genetic means coupled with characterization of their mammalian homologs and (ii) screening for proteins capable of inducing cell death directly in mammalian cells. The Fas antigen/APO-1 is an example of a protein discovered using the latter approach, as it was first discovered as an inducer of cell death and later shown to be necessary and sufficient for certain programmed deaths in vivo. More recent studies have connected Fas to elements of cell death pathways in other species. It has been proposed that Fas is related to the Drosophila cell death protein Reaper, and that in signaling cell death Fas relies upon a relative of the C. elegans cell death protein CED-3. Fas may therefore represent an evolutionarily conserved component of a universal cell death pathway.     

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.     

Monitoring of CD95 (APO-1/Fas) ligand expression in human T cells by quantitative RT-PCR

CD95 (APO-1/Fas) receptor/ligand interaction is a key regulatory pathway for apoptosis in lymphoid cells. We developed a quantitative RT-PCR for the human CD95-L to determine expression levels in lymphoid cell lines and in lymphocytes derived from blood of healthy individuals. In untreated peripheral blood T lymphocytes and T cell lines constitutive expression of the CD95-L mRNA was found at low levels. Stimulation of T cells by treatment with PMA and ionomycine (P/I) lead up to a 100-fold maximal increase in CD95-L mRNA after 4 h. CD95-L mRNA is produced by activated CD8 and CD4T cells. In vivo increased CD95-L mRNA expression was found in freshly isolated T cells during the acute phase of EBV infection. In contrast to T cells, CD95-L mRNA could be induced in some B lineage cell lines only after five days of stimulation. Since defective or accelerated CD95/CD95-L interaction is considered to be involved in the pathogenesis of lymphoproliferation, autoimmunity and AIDS, the quantitative RT-PCR assay described in this paper may provide a powerful tool for monitoring CD95-L expression in these diseases.     

CD95 (APO-1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway

CD95 (APO-1/Fas) is a member of the tumor necrosis factor receptor family, which can trigger apoptosis in a variety of cell types. However, little is known of the mechanisms underlying cell susceptibility to CD95-mediated apoptosis. Here we show that human T cells that are susceptible to CD95-mediated apoptosis, exhibit a constitutive polarized morphology, and that CD95 colocalizes with ezrin at the site of cellular polarization. In fact, CD95 co-immunoprecipitates with ezrin exclusively in lymphoblastoid CD4(+) T cells and primary long-term activated T lymphocytes, which are prone to CD95-mediated apoptosis, but not in short-term activated T lymphocytes, which are refractory to the same stimuli, even expressing equal levels of CD95 on the cell membrane. Pre-treatment with ezrin antisense oligonucleotides specifically protected from the CD95-mediated apoptosis. Moreover, we show that the actin cytoskeleton integrity is essential for this function. These findings strongly suggest that the CD95 cell membrane polarization, through an ezrin-mediated association with the actin cytoskeleton, is a key intracellular mechanism in rendering human T lymphocytes susceptible to the CD95-mediated apoptosis.     

Interferon alpha augments activation-induced T cell death by upregulation of Fas (CD95/APO-1) and Fas ligand expression.

Interferon alpha (IFN-alpha) plays a prominent role in the therapy of a variety of diseases. The Fas/FasL system is crucial for the cytotoxic function and the peripheral elimination of activated T lymphocytes (ATC) by a mechanism referred to as activation-induced cell death (AICD). Recent studies suggest a link between IFN-alpha, the 2', 5'- oligoadenylate system and apoptosis. We therefore asked whether IFN-alpha is able to regulate the Fas/FasL pathway and thereby affects AICD. Peripheral blood mononuclear cells (PBMC), purified T cells and ATC of healthy volunteers were stimulated with various agents and the influence of IFN-alpha on Fas/FasL was assessed by mRNA and protein studies. The proportion of ATC undergoing AICD or anti-Fas-induced apoptosis was determined by FITC-annexin V staining and propidium iodide uptake. IFN-alpha upregulated mRNA expression of Fas and FasL in activated PBMC. Furthermore the concentration of the soluble form of FasL (sFasL) was increased in PBMC and T cells co-stimulated with IFN-alpha and various agents, whereas Fas surface expression was enhanced by IFN-alpha alone. IFN-alpha enhanced apoptosis induced by anti-Fas antibody and augmented AICD via the Fas/FasL pathway. IFN-alpha-regulated AICD may contribute to lymphopenia observed during IFN-alpha therapy. Our data further support that IFN-alpha is a multifunctional cytokine with profound effects on the immune cascades.     

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.     

Protein kinase C inhibits CD95 (Fas/APO-1)-mediated apoptosis by at least two different mechanisms in Jurkat T cells.

We have recently reported that activation of protein kinase C (PKC) plays a negative role in CD95-mediated apoptosis in human T cell lines. Here we present data indicating that although the PKC-induced mitogen-activated protein kinase pathway could be partially implicated in the abrogation of CD95-mediated apoptosis by phorbol esters in Jurkat T cells, the major inhibitory effect is exerted through a PKC-dependent, mitogen-activated protein kinase-independent signaling pathway. Furthermore, we demonstrate that activation of PKC diminishes CD95 receptor aggregation elicited by agonistic CD95 Abs. On the other hand, it has been reported that UV radiation-induced apoptosis is mediated at least in part by the induction of CD95 oligomerization at the cell surface. Here we show that activation of PKC also inhibits UVB light-induced CD95 aggregation and apoptosis in Jurkat T cells. These results reveal a novel mechanism by which T cells may restrain their sensitivity to CD95-induced cell death through PKC-mediated regulation of CD95 receptor oligomerization at the cell membrane.     

Disturbances of the CD95 (APO-1/Fas) system in disorders of lymphohaematopoietic cells

Control of tissue homeostasis is maintained through programs that balance proliferation and cell death. Physiologic cell death is primarily mediated through apoptosis. Deregulations of the cellular programs and genes that determine apoptosis have recently been considered to be involved in a variety of human diseases. One of the central regulatory systems for apoptosis is the CD95 (APO-1/Fas) system. Defects in the CD95 cell surface receptor and deregulated expression of CD95 and the CD95 ligand have been shown to be involved in diseases such as lymphoproliferation, AIDS and haematopoietic failure. This review summarizes our current knowledge on the implication of the CD95 system especially in lymphohaematopoietic diseases in humans.     

Cutting edge: Rac GTPases sensitize activated T cells to die via Fas

In activated CD4(+) T cells, TCR restimulation triggers apoptosis that depends on interactions between the death receptor Fas and its ligand, FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but what pathways mediate this process are not known. In this study we identify the Rho GTPases Rac1 and Rac2 as essential components in restimulation-induced cell death. RNA interference-mediated knockdown of Rac GTPases greatly reduced Fas-dependent, TCR-induced apoptosis. The ability of Rac1 to sensitize T cells to Fas-induced apoptosis correlated with Rac-mediated cytoskeletal reorganization, dephosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal linker proteins, and the translocation of Fas to lipid raft microdomains. In primary activated CD4(+) T cells, Rac1 and Rac2 were independently required for maximal TCR-induced apoptosis. Activating Rac signaling may be a novel way to sensitize chronically stimulated lymphocytes to Fas-induced apoptosis, an important goal in the treatment of autoimmune diseases.     

Ultraviolet Light Induces Apoptosis via Direct Activation of CD95 (Fas/APO-1) Independently of Its Ligand CD95L

Induction of apoptosis in keratinocytes by UV light is a critical event in photocarcinogenesis. Although p53 is of importance in this process, evidence exists that other pathways play a role as well. Therefore, we studied whether the apoptosis-related surface molecule CD95 (Fas/APO-1) is involved. The human keratinocyte cell line HaCaT expresses CD95 and undergoes apoptosis after treatment with UV light or with the ligand of CD95 (CD95L). Incubation with a neutralizing CD95 antibody completely prevented CD95L-induced apoptosis but not UV-induced apoptosis, initially suggesting that the CD95 pathway may not be involved. However, the protease CPP32, a downstream molecule of the CD95 pathway, was activated in UV-exposed HaCaT cells, and UV-induced apoptosis was blocked by the ICE protease inhibitor zVAD, implying that at least similar downstream events are involved in CD95- and UV-induced apoptosis. Activation of CD95 results in recruitment of the Fas-associated protein with death domain (FADD) that activates ICE proteases. Immunoprecipitation of UV-exposed HaCaT cells revealed that UV light also induces recruitment of FADD to CD95. Since neutralizing anti-CD95 antibodies failed to prevent UV-induced apoptosis, this suggested that UV light directly activates CD95 independently of the ligand CD95L. Confocal laser scanning microscopy showed that UV light induced clustering of CD95 in the same fashion as CD95L. Prevention of UV-induced CD95 clustering by irradiating cells at 10°C was associated with a significantly reduced death rate. Together, these data indicate that UV light directly stimulates CD95 and thereby activates the CD95 pathway to induce apoptosis independently of the natural ligand CD95L. These findings further support the concept that UV light can affect targets at the plasma membrane, thereby even inducing apoptosis.     

Early detachment of colon carcinoma cells during CD95(APO-1/Fas)- mediated apoptosis. I. De-adhesion from hyaluronate by shedding of CD44

Ligation of CD95 (APO-1/Fas) cell surface receptors induces death in apoptosis-sensitive cells. Induction of apoptosis in adherent gamma interferon-stimulated HT-29 and COLO 205 colon carcinoma cells by cross- linking CD95 with anti-APO-1 monoclonal antibody resulted in detachment of the cells from hyaluronate starting about 1 h after antibody exposure. Loss of adhesion was paralleled by a substantial reduction of the multifunctional cell surface adhesion molecule CD44. As evidenced by cycloheximide treatment, this effect was not caused by impaired protein synthesis. Depletion of surface CD44 was also not due to membrane blebbing, since cytochalasin B failed to inhibit ascension from hyaluronate. Instead, ELISA and time kinetics showed increasing amounts of soluble CD44 in the supernatant of CD95-triggered cells. SDS- PAGE revealed that soluble CD44 had an apparent molecular mass of about 20 kD less than CD44 immunoprecipitated from intact cells. Thus, CD95- triggering induced shedding of CD44. Shedding is a novel mechanism operative in early steps of CD95-mediated apoptosis. Shedding surface molecules like CD44 might contribute to the active disintegration of dying epithelial cells in vivo.     

Cutting edge: soluble HLA-G1 triggers CD95/CD95 ligand-mediated apoptosis in activated CD8+ cells by interacting with CD8

The nonpolymorphic soluble HLA-G1 (sHLA-G1) isoform has been reported to be secreted by trophoblast cells at the materno-fetal interface, suggesting that it may act as immunomodulator during pregnancy. In this paper, we report that affinity-purified beta2-microglobulin-associated sHLA-G1 triggered apoptosis in activated, but not resting CD8+ peripheral blood cells. We demonstrate by Western blotting that sHLA-G1 enhanced CD95 ligand expression in activated CD8+ cells. Cytotoxicity was inhibited by preincubation of the cells with a CD95 antagonist mAb (ZB4) or a soluble recombinant CD95-Fc, indicating that apoptosis is mediated through the CD95/CD95 ligand pathway. Finally, we show that such sHLA-G1-induced apoptosis depends on the interaction with CD8 molecules, with cell death being blocked by various CD8 mAbs.