CD95-mediated Apoptosis Research Articles

Regulation of CD95/APO-1/Fas-induced apoptosis by protein phosphatases

Triggering the CD95/APO-1/Fas receptor by CD95-L induces the assembly of the death-inducing signaling complex (DISC), which permits initiator caspases activation and progression of a signaling cascade that culminates in cellular apoptosis. Despite the CD95 receptor does not exhibit any kinase activity by itself, phosphorylation/dephosphorylation events seem important to regulate many aspects of CD95-mediated apoptosis. Here, we try to highlight particularly the importance of protein phosphatases in the modulation of the CD95 system.

Human leukocyte elastase counteracts matrix metalloproteinase-7 induced apoptosis resistance of tumor cells

Matrix metalloproteinase-7 (MMP-7/Matrilysin) is a component of the tumor microenvironment associated with malignant progression. Its expression in tumors protects tumor cells from CD95-mediated apoptosis and the cytotoxic activity of tumor specific CD8 + T cells. In the present study, we show that human leukocyte elastase (HLE) secreted by polymorphonuclear leukocytes cleaves MMP-7 resulting in loss of enzymatic activity. The anti-apoptotic effect of MMP-7 is reduced in the presence of HLE for CD95-, doxorubicin- and CTL-mediated apoptosis. Our data indicates that HLE may be a natural inactivator of MMP-7 which can counteract MMP-7-induced apoptosis resistance.

Yes and PI3K Bind CD95 to Signal Invasion of Glioblastoma

Invasion of surrounding brain tissue by isolated tumor cells represents one of the main obstacles to a curative therapy of glioblastoma multiforme. Here we unravel a mechanism regulating glioma infiltration. Tumor interaction with the surrounding brain tissue induces CD95 Ligand expression. Binding of CD95 Ligand to CD95 on glioblastoma cells recruits the Src family member Yes and the p85 subunit of phosphatidylinositol 3-kinase to CD95, which signal invasion via the glycogen synthase kinase 3-beta pathway and subsequent expression of matrix metalloproteinases. In a murine syngeneic model of intracranial GBM, neutralization of CD95 activity dramatically reduced the number of invading cells. Our results uncover CD95 as an activator of PI3K and, most importantly, as a crucial trigger of basal invasion of glioblastoma in vivo.

Construction of Optimized Bispecific Antibodies for Selective Activation of the Death Receptor CD95

We have previously reported that bispecific antibodies directed to different target antigens on lymphoma cells and to the death receptor CD95/Fas/Apo-1 selectively kill these cells, thus providing an attractive strategy for the selective stimulation of CD95 on the surface of tumor cells. Here, we further explore the general applicability of this approach under more stringent conditions using various bispecific antibodies directed to different target antigens on glioblastoma cells which express relatively low levels of CD95. We found that bispecific CD95 antibodies targeting the neuronal glial antigen-2 induce CD95-mediated apoptosis selectively in glioblastoma cells expressing this target antigen. A recombinant bispecific single-chain antibody was as effective as a chemically hybridized F(ab')(2) fragment with identical specificities. In contrast, a bispecific F(ab')(2) fragment binding to the epidermal growth factor receptor on the glioblastoma cells failed to induce apoptosis. This is most likely due to the exclusively unicellular binding of this particular fragment to target cells expressing both the epidermal growth factor receptor and CD95. If this type of binding in a cis configuration is favored by a particular bispecific antibody, rather than a bicellular binding in trans, effective cross-linking of CD95 does not occur and apoptosis is not induced. To facilitate bicellular binding in a trans configuration, we constructed a bispecific antibody directed to the extracellular matrix protein tenascin. As expected, this reagent was the most effective of all the antibodies tested. The presence of sensitizing reagents such as cycloheximide and various cytostatic drugs further enhanced antibody-mediated killing of the tumor cells. We believe that these results may point the way to a successful application of bispecific CD95 antibodies in experimental tumor therapy.

Anti-apoptotic and growth-stimulatory functions of CK1 delta and epsilon in ductal adenocarcinoma of the pancreas are inhibited by IC261 in vitro and in vivo

Background: Pancreatic ductal adenocarcinomas (PDACs) are highly resistant to treatment due to changes in various signalling pathways. CK1 isoforms play important regulatory roles in these pathways. Aims: We analysed the...

Genetic aspects of pediatric asthma: potential clinical role

Background: Beta cell apoptosis has been associated with insulin dependent diabetes mellitus (IDDM) onset in newly diagnosed diabetic patients. There is an emerging evidence that T cell-induced apoptosis is a dominant effector mechanism in diabetes mellitus type 1 (DM1). Pancreatic β-cells derived from newly diagnosed type 1 diabetics were found to have increased cell surface expression of Fas (CD95) compared to β-cells from healthy subjects. Objective: The study investigates the spontaneous lymphocyte apoptosis via CD95 molecule expression to demonstrate activation induced cell death in children with high risk of DM1 and in type 1 diabetics under insulin therapy. Methods: This study comprised 90 children and adolescents, divided into 3 groups. G(1) comprised 40 type-1 diabetics, their ages ranging from 8.0 to 17.0 years and disease duration between 2.0 and 12.0 years. G(2) (prediabetics) included 30 euglycaemic subjects who were first degree relatives of type 1 diabetics, with normal fasting blood glucose and positive first phase insulin release (FPIR) and/or positive islet cell (ICA) or glutamic acid decarboxylase (GAD) antibodies. G(3) comprised 20 healthy, age and sex matched subjects with no clinical or laboratory signs or family history of type-1DM. Patients were subjected to clinical evaluation with special emphasis on signs suggestive of microvascular complications. The study measurements included random blood sugar (RBS), glycosylated hemoglobin (HbA 1c ), urinary microalbumin assay and flow cytometric assessment of apoptosis by measuring CD95 percentage expression on CD3 lymphocytes. Results: The percentage of CD95 positive T-lymphocytes was significantly higher in prediabetics than in type-1 diabetics and controls (57.687±6.68, 45.01±6.648,16.75±4.98% respectively; p < 0.001). CD3 positive lymphocytes were significantly lower in prediabetics than type-1 diabetics and controls (52.93±11.64, 66.23±7.04, 63.910±3.4% respectively; p < 0.001). The percentage of CD95 on T-lymphocytes could not be correlated with age, insulin dose and RBS, but HbA1c was positively correlated with both CD3 lymphocytes and CD95% expression. Complicated type-1 diabetics showed higher CD95% expression compared to noncomplicated patients. Conclusion: Peripheral blood lymphocytes with CD95 antigen expression are increased in prediabetics. As CD95 is an important receptor for activation-induced cell death, CD95 mediated apoptosis could play a potential role in the pathogenesis of DM1. Keywords: lymphocyte apoptosis; CD95 system; type 1 DM; prediabetes Egypt J Pediatr Allergy Immunol 2008; 6(2): 57-67

CD95-mediated apoptosis in naïve, central and effector memory subsets of CD4+ and CD8+ T cells in aged humans

Aging is associated with a decrease in naïve (T N) and central memory (T CM), and an accumulation of effector memory (T EM and T EMRA) T cell subsets. Previously, we have demonstrated an increased sensitivity of T N and T CM CD4+ and CD8+ T cells in aging to TNF-α-induced apoptosis. In this investigation, we examined whether similar differential sensitivity is applicable to CD95-mediated apoptosis. We show that T N and T CM CD4+ and CD8+ T cells from aged subjects are significantly more sensitive to CD95-mediated apoptosis. Increased apoptosis is associated with increased activation of caspase-8 and caspase-3. Both caspase-8 and caspase-3 inhibitors blocked CD95-mediated apoptosis and activation of caspase-8 and caspase-3 in T N and T CM CD4+ and CD8+ T cells. No significant difference was observed in apoptosis or in activation of caspase-8 and caspase-3 in T EM and T EMRA CD4+ and CD8+ T cells between young and aged subjects; both populations were relatively and comparably resistant to CD95-mediated apoptosis and caspase activation. No correlation was observed between the sensitivity/resistance of any of the subsets of CD4+ or CD8+T cells to CD95-mediated apoptosis and the expression of CD95. Our data suggest that increased CD95-mediated apoptosis of T N and T CM CD8+ and CD4+ T cells may play a role in their decline in human aging.

Paclitaxel (Taxol®)-induced apoptosis in human epithelioid sarcoma cell lines is enhanced by upregulation of CD95 ligand (FasL/Apo-1L)

Metastasizing epithelioid sarcoma (ES) is an extremely aggressive tumor, because conventional chemotherapy and irradiation are largely ineffective. Here, we analyzed the impact of the CD95-mediated drug-induced apoptosis in ES cell lines. The effects of paclitaxel (Taxol) and 5-FU were determined by MTT assay. The extent of apoptosis was analyzed by light microscopy and Annexin V staining (flow cytometry). The expression of death receptors and ligands was defined by RT-PCR, Western blotting and flow cytometry. All cell lines expressed CD95, but not the CD95 ligand. The CD95 activation resulted in apoptosis and cell death in all cell lines. Both paclitaxel and 5-FU are able to trigger apoptosis, and furthermore, to upregulate CD95, whereas only paclitaxel increases CD95 ligand expression. Neutralizing antibodies directed against CD95 ligand effectively inhibited paclitaxel-induced cell death, thereby providing evidence for a direct involvement of the CD95 system in paclitaxel-induced apoptosis. Concomitant upregulation of CD95 receptor and ligand may significantly enhance the response of ES to anticancer drugs. As evident from the differential response of our clonal ES subpopulations to paclitaxel and 5-FU, effective activation of the CD95 system depends on intrinsic properties of both the chemotherapeutic agent and target cell population.

Conversion of CD95 (Fas) Type II into Type I signaling by sub-lethal doses of cycloheximide

CD95 (Fas/Apo-1)-mediated apoptosis was shown to occur through two distinct pathways. One involves a direct activation of caspase-3 by large amounts of caspase-8 generated at the DISC (Type I cells). The other is related to the cleavage of Bid by low concentration of caspase-8, leading to the release of cytochrome c from mitochondria and the activation of caspase-3 by the cytochrome c/APAF-1/caspase-9 apoptosome (Type II cells). It is also known that the protein synthesis inhibitor cycloheximide (CHX) sensitizes Type I cells to CD95-mediated apoptosis, but it remains contradictory whether this effect also occurs in Type II cells. Here, we show that sub-lethal doses of CHX render both Type I and Type II cells sensitive to the apoptogenic effect of anti-CD95 antibodies but not to chemotherapeutic drugs. Moreover, Bcl-2-positive Type II cells become strongly sensitive to CD95-mediated apoptosis by the addition of CHX to the cell culture. This is not the result of a restraint of the anti-apoptotic effect of Bcl-2 at the mitochondrial level since CHX-treated Type II cells still retain their resistance to chemotherapeutic drugs. Therefore, CHX treatment is granting the CD95-mediated pathway the ability to bypass the mitochondria requirement to apoptosis, much alike to what is observed in Type I cells.

CD95 signaling deficient mice with a wild-type hematopoietic system are prone to hepatic neoplasia

Patients with mutations in the death receptor CD95 (Fas/APO-1) frequently develop B-cell lymphoma. However, solid tumors have not been found in the context of defective CD95. This could be due to the fatal autoimmune proliferative disease that develops in the absence of functional CD95 or to a difference in CD95 signaling in lymphoid versus nonlymphoid tissues. To test this we reconstituted mice that harbor a point mutation in the death domain of CD95 (lpr(cg) mice), either in one or in both alleles, with bone marrow from wild-type (wt) mice. After a year one third of the lpr(cg)/lpr(cg) mice developed spontaneous hepatic neoplasms. In contrast only one of the wt/lpr(cg) mice and none of the wt mice developed liver cancer. The agonistic anti-CD95 antibody Jo2 induced massive apoptosis in the liver of wt mice but not in the livers of either wt/lpr(cg) or lpr(cg)/lpr(cg) mice. The susceptibility of lpr(cg)/lpr(cg) mice to liver cancer cannot solely be due to impaired CD95 mediated apoptosis because there was no clear correlation between apoptosis resistance and tumor formation. A gene chip analysis identified genes selectively upregulated in the liver of wt and wt/lpr(cg) mice which may protect these mice from developing liver cancer. Our data represent the first case of CD95 protecting from developing a solid cancer.

Up-regulation of c-FLIPS+R upon CD40 stimulation is associated with inhibition of CD95-induced apoptosis in primary precursor B-ALL

Previous studies on apoptosis defects in acute lymphoblastic leukemia (ALL) have focused on chemotherapy-induced, primarily mitochondrial death pathways. Yet, immunologic surveillance mechanisms including sensitization to apoptotic signals mediated via the death receptor CD95 might contribute to leukemic control. Here, we show that primary B-cell precursor ALL cells from children escape from receptor-dependent cell death in 2 ways: Resting ALL blasts are protected from receptor-mediated apoptosis due to the absence of CD95 surface expression. However, even though CD40 ligation results in up-regulation of CD95, ALL blasts, unlike normal B cells, remain resistant to apoptosis. We show that this apoptosis resistance involves the selective up-regulation of the short isoforms of the caspase-8 inhibitor c-FLIP acting directly at the CD95 receptor level. Treatment with cycloheximide during CD40 activation prevents up-regulation of those c-FLIP isoforms and sensitizes ALL cells toward CD95-mediated apoptosis. We therefore propose that induction of the short c-FLIP isoforms inhibits the onset of CD95-induced apoptosis in primary CD40-stimulated ALL cells despite high CD95 expression.

NRAS mutation causes a human autoimmune lymphoproliferative syndrome

The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization, and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous, and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4(-), CD8(-) alphabeta T cells. Mutations in ALPS typically affect CD95 (Fas/APO-1)-mediated apoptosis, one of the extrinsic death pathways involving TNF receptor superfamily proteins, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair CD95-mediated apoptosis. The increase in active, GTP-bound NRAS augments RAF/MEK/ERK signaling, which markedly decreases the proapoptotic protein BIM and attenuates intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects. Our observations on the effects of NRAS activation indicate that RAS-inactivating drugs, such as farnesyltransferase inhibitors should be examined in human autoimmune and lymphocyte homeostasis disorders.

Analysis of CD95 Threshold Signaling: TRIGGERING OF CD95 (FAS/APO-1) AT LOW CONCENTRATIONS PRIMARILY RESULTS IN SURVIVAL SIGNALING

Recently we generated a mathematical model (Bentele, M., Lavrik, I., Ulrich, M., Stosser, S., Heermann, D. W., Kalthoff, H., Krammer, P. H., and Eils, R. (2004) J. Cell Biol. 166, 839-851) of signaling in CD95(Fas/APO-1)-mediated apoptosis. Mathematical modeling in combination with experimental data provided new insights into CD95-mediated apoptosis and allowed us to establish a threshold mechanism of life and death. Here, we further assessed the predictability of the model experimentally by a detailed analysis of the threshold behavior of CD95 signaling. Using the model predictions for the mechanism of the threshold behavior we found that the CD95 DISC (death-inducing signaling complex) is formed at the cell membrane upon stimulation with low concentrations of agonistic anti-APO-1 monoclonal antibodies; however, activation of procaspase-8 at the DISC is blocked due to high cellular FLICE-inhibitory protein recruitment into the DISC. Given that death signaling does not occur upon CD95 stimulation at low (threshold) anti-APO-1 concentrations, we also analyzed survival signaling, focusing on mitogen-activated protein kinase activation. Interestingly, we found that mitogen-activated protein kinase activation takes place under threshold conditions. These findings show that triggering of CD95 can signal both life or death, depending on the strength of the stimulus.

Sensitization of p53-mutated epithelial ovarian cancer to CD95-mediated apoptosis is synergistically induced by cisplatin pretreatment

Epithelial ovarian carcinoma (EOC) remains a highly lethal malignancy. Despite the progress in surgical and therapeutic strategies, resistance to chemotherapy is still a major concern. Cytotoxic therapies mediate killing of cancer cells by activating the intrinsic mitochondrial apoptotic pathway, and p53 status is a key factor in determining the efficacy of apoptotic signaling. The extrinsic (CD95) death receptor-dependent signaling pathway also contributes to the efficacy of cancer therapy. We previously showed that EOC are generally resistant to CD95-dependent apoptosis. In p53 wild-type EOC tumors, CD95-mediated apoptosis is impaired at the receptor level by the long form of cellular FLICE-inhibitory protein, whereas this mechanism does not account for resistance in tumors with mutated p53 (p53mu). In the present study, we examined both intrinsic and death receptor-dependent apoptotic signaling in p53mu OVCAR3 EOC cell line, showing that these cells are less susceptible to cisplatin treatment as compared with p53 wild-type EOC cells and also resist CD95-mediated apoptosis due to inefficient formation of the death-inducing signaling complex and weak mitochondrial signal amplification. However, pretreatment of OVCAR3 cells with clinically relevant cisplatin concentrations significantly improved receptor-dependent apoptotic signaling by up-modulating CD95 receptor expression and increasing death-inducing signaling complex formation efficiency. The synergy of cisplatin pretreatment and CD95 triggering in inducing cell death was also shown in p53mu tumor cells derived from ascitic fluid of advanced-stage EOC patients. These findings support the effectiveness of a combined therapeutic treatment able to sensitize cancer cells to apoptosis even when p53 is functionally inactivated.

Interleukin-4 Inhibits Caspase-3 by Regulating Several Proteins in the Fas Pathway during Initial Stages of Human T Helper 2 Cell Differentiation

Interleukin-4 (IL-4) is the main cytokine that polarizes activated naïve CD4+ T cells in the T helper 2 (Th2) direction. IL-4 also regulates the subsequent stages of Th2 cell-mediated diseases, such as allergies. We conducted a proteomics study to identify IL-4-induced differences during the initial stages of T helper cell differentiation. Primary CD4+ T lymphocytes were isolated from human cord blood, activated through CD3 and CD28, and cultured in the presence or absence of IL-4. Soluble proteins were separated by two-dimensional electrophoresis and visualized by staining with autoradiography, which indicated that at least 20 proteins might be regulated by IL-4. From this minimum of 20 stained proteins, altogether 35 proteins were identified using tandem mass spectrometry. Interestingly the fragmented form of GDP dissociation inhibitor expressed in lymphocytes/Rho GDP dissociation inhibitor 2 (Ly-GDI), a known target of Caspase-3, was observed to be down-regulated in IL-4-treated cells. It was shown in further studies that IL-4 decreases Caspase-3 activity and cell death in these cells. Neutralizing Fas-Fas ligand interaction led to decreased Caspase-3 activity and lowered Ly-GDI fragmentation. We further characterized the effects of IL-4 on the expression of main regulators in the Fas-mediated pathway. We demonstrated that IL-4 decreases expression of Fas receptor and increases expression of Bid, Bcl-2, and Bcl-xL. Importantly IL-4 significantly up-regulated the short form of c-FLIP, although the levels of c-FLIP long were unaltered after IL-4 induction. Taken together, our results indicate that IL-4 inhibits caspase activity during the initial stages of human Th2 cell differentiation by regulating expression of several key players in the Fas-induced pathway.

Enhanced CD95-mediated apoptosis contributes to radiation hypersensitivity of NBS lymphoblasts

The molecular causes for enhanced radiosensitivity of Nijmegen Breakage Syndrome cells are unclear, especially as repair of DNA damage is hardly impeded in these cells. We clearly demonstrate that radiation hypersensitivity is accompanied by enhanced gamma-radiation-induced apoptosis in NBS1 deficient lymphoblastoid cell lines. Differences in the apoptotic behavior of NBS1 (-/-) and NBS1 (+/-) cells are not due to an altered p53 stabilization or phosphorylation in NBS1 (-/-) cells. gamma-radiation-induced caspase-8 activity is increased and visualization of CD95 clustering by laser scanning microscopy shows a significant higher activation of the death receptor in NBS1 (-/-) cells. Further investigation of the molecular mechanisms reveals a role for reactive oxygen species-triggered activation of CD95. These results demonstrate that NBS1 suppresses the CD95 death receptor-dependent apoptotic pathway after gamma-irradiation and evidence is given that this is achieved by regulation of the PI3-K/AKT survival pathway.

Mitochondrial function in liver disease

Oxidative stress is involved in the pathogenesis and progression of different liver diseases, such as alcoholic liver disease and biliary cirrhosis. The increased mitochondrial production of O2(-) at complexes I and III, and consequently of H2O2 and other reactive oxygen species (ROS), triggered by NADH overproduction seems the major cause of mitochondrial and cellular oxidative stress and damage in chronic alcoholism. The mitochondrial oxidative stress renders hepatocytes susceptible to ethanol- or acetaldehyde-induced mitochondrial membrane permeability transition (MMPT) and apoptosis. Nitrosative stress contributes to cell death by peroxynitrite formation. The expression of the death receptor ligand CD95 is also up-regulated by acetaldehyde metabolism. Consequently, a dual mechanism, NADH-driven MMPT and CD95-mediated apoptosis, involving in both cases acetaldehyde metabolism and ROS production, operates in ethanol-induced apoptosis. In the biliary cirrhosis induced by chronic cholestasis, liver mitochondria show increased H2O2 production and GSH depletion and oxidation. Dysfunctional hepatocytes, with a loss in mitochondrial cardiolipin and decreased mitochondrial membrane potential evolve during cholestasis to apoptosis. Ursodeoxycholic acid prevents enlargement of this population as well as mitochondrial oxidative stress. Mitochondrial oxidative stress precedes the initiation and execution of hepatocyte apoptosis in chronic alcoholism and biliary cirrhosis. We suggest that overproduction of mitochondrial NADH is the primary cause for the development of alcoholic and non-alcoholic liver disease by a situation of chronic mitochondrial oxidative stress, which should be considered the second hit that renders hepatocytes susceptible to cell injury and apoptosis.

Serum levels of soluble CD95 are not associated with amelioration of multiple sclerosis during pregnancy

Multiple sclerosis (MS) is considered an autoimmune disease of the central nervous system. Like various other autoimmune disorders, MS normally improves during pregnancy. Pregnant MS patients experience a significant reduction in relapse rates and magnetic resonance (MR) disease activity. How sex steroid hormones affect disease course remains unclear. We hypothesized that hormonal changes during pregnancy might modulate the autoimmune response by enhancing apoptosis of autoreactive T lymphocytes. One of the most important effectors of apoptosis in T cells is the CD95/CD95L system. We have previously reported that the soluble form of CD95 (sCD95) can block CD95-mediated apoptosis and that MS patients show elevated levels of sCD95. Therefore, we considered whether gravidity might influence serum levels of sCD95 in patients, and analyzed the concentration of sCD95 in the sera of 61 patients with relapsing-remitting (RR) MS before, during and after pregnancy. We found no association between serum levels of sCD95 and pregnancy-related immune suppression in MS patients. Thus, sex steroid hormones do not seem to affect the production of anti-apoptotic sCD95.

HIV-1 Tat and T cell apoptosis

CD95, a member of the tumor necrosis factor (TNF) receptor superfamily induces apoptosis upon receptor oligomerization. The receptor and its ligand are important for apoptosis of peripheral T cells, for downregulation of an immune response and most likely, at least in part, also for peripheral T cell tolerance. In AIDs, apoptosis mediated by this system might contribute to the depletion of T helper lymphocytes. Likewise, in diseases in which liver cells are destroyed the CD95 system might play a major role. In a search to identify the intracellular signalling pathway of CD95 several molecules coupling to oligomerized CD95 were immunoprecipitated from apoptosis-sensitive human leukemic T cell and lymphoblastoid B cell lines. The following binding molecules were only associated with aggregated and not with monomeric CD95: phosphorylated FADD (MORT1) and caspase 8. Thus, caspase 8 was identified as the most CD95 receptor proximal protease which starts the cascade of protease reactions important for CD95-mediated apoptosis. Association of FADD and caspase 8 with CD95 was not observed with C-terminally truncated non-signalling CD95. FADD and FLICE did also not associate with a CD95 cytoplasmic tail carrying the lprcg amino acid replacement. FADD and caspase 8 form a death-inducing signalling complex (DISC) with the CD95 receptor and are, thus, the first CD95 associating proteins of a signalling cascade mediating apoptosis.The function of the DISC is discussed in detail, particularly with respect to its role in sensitivity and resistance to apoptosis. The CD95 death system plays a role in destruction of liver tissue. In hepatitis cytotoxic T lymphocytes might use the CD95 system to kill infected hepatocytes. In M. Wilson copper overload leads to upregulation of the CD95 ligand that may finally contribute to acute liver failure. In HCC from patients treated with chemotherapeutic drugs the CD95 receptor and ligand are upregulated and may contribute to apoptosis of the tumor or, dependent on the drug sensitivity of the tumor, to the status of the tumor as an immunoprivileged site. In AIDS, HIV Tat made by virus infected cells enters noninfected cells and causes CD95 mediated apoptosis by upregulating the CD95 ligand. This might be a cause for increased T cell depletion in this disease. [1,2].

Cyclosporin A Abolishes CD28-Mediated Resistance to CD95-Induced Apoptosis via Superinduction of Caspase-3

Costimulation of T cells via CD28 promotes both proliferation and resistance to apoptosis. In this study, we show that the immunosuppressive drug cyclosporin A (CsA) fully reverses resistance to CD95-mediated cell death after TCR/CD28 costimulation or superagonistic anti-CD28 mAb stimulation of primary rat lymph node T cells. This effect correlated with a pronounced superinduction of caspase-3 on both mRNA and protein levels, whereas its main antagonist, X chromosome-linked inhibitor of apoptosis, was unaffected by inclusion of CsA. Apoptosis triggered by CD95 cross-linking was characterized by robust caspase-3 activation. Furthermore, CsA sensitization to CD95-mediated apoptosis of CD28-activated T cells did not alter mRNA stability of superinduced caspase-3 mRNA, suggesting a transcriptional regulation of the caspase-3 gene. Addition of Ca(2+) ionophores to TCR/CD28 or superagonistic CD28-stimulated cells reduced caspase-3 levels, further supporting a role for Ca(2+)-dependent signaling pathways in negatively regulating caspase-3. Taken together, these findings suggest that CsA promotes sensitivity to CD95-mediated apoptosis in CD28-stimulated T cells by superinduction of the caspase-3 gene via a mechanism involving suppression of the calcineurin pathway.