Another cell death induction system: TNF-alpha acts as a ligand for Fas in vaginal cells.

The death receptor Fas transduces apoptotic death signaling upon stimulation with the Fas ligand. We previously reported that Fas contributes to vaginal cell death observed during the estrus cycle and after estrogen deprivation, using the functional Fas-lacking lpr and lprcg mutant mouse. In the present study, we investigated whether the Fas ligand also plays a dominant role in vaginal cell death using the functional Fas ligand-lacking gld mutant mouse. Our results demonstrated that vaginal cells of gld mice do not show any abnormalities, suggesting the possible presence of another ligand for Fas. Through our investigation, we demonstrated TNF-alpha as a ligand for vaginal Fas. Here, we propose that TNF-alpha acts for the ligand for Fas in vaginal cells, suggesting a new cell death induction system.     

Apoptosis induced by TGF-beta 1 in Burkitt's lymphoma cells is caspase 8 dependent but is death receptor independent

TGF-beta is a potent inducer of apoptosis in many Burkitt's lymphoma (BL) cell lines. In this study, we characterize this apoptotic process in the EBV-negative BL41 cell line. Induction of apoptosis was detected as early as 8 h after TGF-beta treatment, as assayed by TUNEL and poly(ADP-ribose) polymerase cleavage. FACS analysis demonstrates that this proceeds predominately from the G1, but also from the G2/M phases of the cell cycle. We observed no early detectable changes in the steady-state levels of Bcl-2 and several of its family members after TGF-beta treatment. We detected cleavage of caspases 2, 3, 7, 8, and 9 into their active subunits. Consistent with the involvement of these enzymes in TGF-beta-mediated apoptosis, the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(Ome)-flouromethylketone (ZVAD-fmk) blocked TGF-beta-induced apoptosis and revealed a G1 arrest in treated cells. Use of specific caspase inhibitors revealed that the induction of apoptosis is caspase 8 dependent, but caspase 3 independent. Activation of caspase 8 has been shown to be a critical event in death receptor-mediated apoptosis. However, TGF-beta treatment of BL41 cells was found not to affect the cell surface expression of Fas, TNF-R1, DR3, DR4, or DR5, or the steady-state expression levels of Fas ligand, TNF-R1, DR3, DR4, and DR5. Furthermore, blocking experiments indicated that TGF-beta-mediated apoptosis is not dependent on Fas ligand, TNF-alpha, tumor necrosis-like apoptosis-inducing ligand, or TNF-like weak inducer of apoptosis signaling. Therefore, it appears that TGF-beta induces apoptosis in BL cell lines via caspase 8 in a death receptor-independent fashion.     

Stimulation of Kv1.3 potassium channels by death receptors during apoptosis in Jurkat T lymphocytes

The loss of intracellular potassium is a pivotal step in the induction of apoptosis but the mechanisms underlying this response are poorly understood. Here we report caspase-dependent stimulation of potassium channels by the Fas receptor in a human Jurkat T cell line. Receptor activation with Fas ligand for 30 min increased the amplitude of voltage-activated potassium currents 2-fold on average. This produces a sustained outward current, approximately 10 pA, at physiological membrane potentials during Fas ligand-induced apoptosis. Both basal and Fas ligand-induced currents were blocked completely by toxins that selectively inhibit Kv1.3 potassium channels. Kv1.3 stimulation required the expression of Fas-associated death domain protein and activation of caspase 8, but did not require activation of caspase 3 or protein synthesis. Furthermore, Kv1.3 stimulation by Fas ligand was prevented by chronic stimulation of protein kinase C with 20 nm phorbol 12-myristate 13-acetate during Fas ligand treatment, which also blocks apoptosis. Thus, Fas ligand increases Kv1.3 channel activity through the same canonical apoptotic signaling cascade that is required for potassium efflux, cell shrinkage, and apoptosis.     

Fas ligand-independent, FADD-mediated activation of the Fas death pathway by anticancer drugs

Trimerization of the Fas receptor (CD95, APO-1), a membrane bound protein, triggers cell death by apoptosis. The main death pathway activated by Fas receptor involves the adaptor protein FADD (for Fas-associated death domain) that connects Fas receptor to the caspase cascade. Anticancer drugs have been shown to enhance both Fas receptor and Fas ligand expression on tumor cells. The contribution of Fas ligand-Fas receptor interactions to the cytotoxic activity of these drugs remains controversial. Here, we show that neither the antagonistic anti-Fas antibody ZB4 nor the Fas-IgG molecule inhibit drug-induced apoptosis in three different cell lines. The expression of Fas ligand on the plasma membrane, which is identified in untreated U937 human leukemic cells but remains undetectable in untreated HT29 and HCT116 human colon cancer cell lines, is not modified by exposure to various cytotoxic agents. These drugs induce the clustering of Fas receptor, as observed by confocal laser scanning microscopy, and its interaction with FADD, as demonstrated by co-immunoprecipitation. Overexpression of FADD by stable transfection sensitizes tumor cells to drug-induced cell death and cytotoxicity, whereas down-regulation of FADD by transient transfection of an antisense construct decreases tumor cell sensitivity to drug-induced apoptosis. These results were confirmed by transient transfection of constructs encoding either a FADD dominant negative mutant or MC159 or E8 viral proteins that inhibit the FADD/caspase-8 pathway. These results suggest that drug-induced cell death involves the Fas/FADD pathway in a Fas ligand-independent fashion.     

Expression of high levels of human proteinase inhibitor 9 blocks both perforin/granzyme and Fas/Fas ligand-mediated cytotoxicity

Proteinase inhibitor 9 (PI-9, SerpinB9) is the only known human intracellular granzyme B inhibitor. Whether expression of PI-9 is sufficient to block cytolysis induced by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells remains controversial. To evaluate the roles of PI-9, we isolated and tested three lines of stably transfected HeLa cells expressing wild-type PI-9 and one line expressing an inactive mutant PI-9. Expressions of wild-type PI-9, but not the inactive mutant PI-9, inhibited cytolysis induced by human NK92 and NKL natural killer cells. Expression of high levels of PI-9 is therefore sufficient to protect human cells against NK cell-mediated cell death. Using two assays, we show that expressing wild-type PI-9, but not the inactive mutant PI-9, blocks Fas/Fas ligand (Fas/FasL)-mediated apoptosis. PI-9 expression has no effect on etoposide-induced apoptosis. HeLa cells exhibiting substantial resistance to Fas/FasL-mediated apoptosis contain 2- to 3-fold higher PI-9 levels than HCT116 human colon cancer cells and 2- to 3-fold lower PI-9 levels than MCF7/ERHA breast cancer cells, in which PI-9 is strongly induced by estrogens, and by tamoxifen. Expression of increasing levels of PI-9 in target cells may progressively inhibit immune surveillance by blocking NK and CTL-induced cytotoxicity through the perforin/granzyme pathway and then through the Fas/FasL pathway.     

Cell death induced by the Fas/Fas ligand pathway and its role in pathology.

Engagement of the cell death surface receptor Fas by Fas ligand (FasL) results in apoptotic cell death, mediated by caspase activation. Cell death mediated via Fas/FasL interaction is important for homeostasis of cells in the immune system and for maintaining immune-privileged sites in the body. Killing via the Fas/FasL pathway also constitutes an important pathway of killing for cytotoxic T cells. Fas ligand is induced in activated T cells, resulting in activation-induced cell death by the Fas/FasL pathway. Recently it has been shown that the Fas receptor can also be up-regulated following a lesion to the cell, particularly that induced by DNA-damaging agents. This can then result in killing of the cell by a Fas/FasL-dependent pathway. Up-regulation of Fas receptor following DNA damage appears to be p53 dependent.     

Inhibition of cell growth and induction of apoptotic cell death by the human tumor-associated antigen RCAS1.

Tumor-associated antigens that can be recognized by the immune system include the MAGE-family, p53, MUC-1, HER2/neu and p21ras. Despite their expression of these distinct antigens, tumor elimination by the immune system is often inefficient. Postulated mechanisms include insufficient expression of co-stimulatory or adhesion molecules by tumor cells, or defective processing and presentation of antigens on their cell surfaces. Tumor cells may also evade immune attack by expressing CD95 (APO-1/Fas) ligand or other molecules that induce apoptosis in activated T cells. Here we describe RCAS1 (receptor-binding cancer antigen expressed on SiSo cells), a membrane molecule expressed on human cancer cells. RCAS1 acts as a ligand for a putative receptor present on various human cell lines and normal peripheral lymphocytes such as T, B and NK cells. The receptor expression was enhanced by activation of the lymphocytes. RCAS1 inhibited the in vitro growth of receptor-expressing cells and induced apoptotic cell death. Given these results, tumor cells may evade immune surveillance by expression of RCAS1, which would suppress clonal expansion and induce apoptosis in RCAS1 receptor-positive immune cells.     

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Induces Caspase-Dependent Interleukin-8 Expression and Apoptosis in Human Astroglioma Cells

Among the tumor necrosis factor (TNF) family of cytokines, FasL and TNF-related apoptosis-inducing ligand (TRAIL) are known to induce cell death via caspase activation. Recently, other biological functions of these death ligands have been postulated in vitro and in vivo. It was previously shown that Fas ligation induces chemokine expression in human glioma cells. In this study, we investigated whether the TRAIL-DR5 system transduces signals similar to those induced by other TNF family ligands and receptors. To address this issue, two human glioma cell lines, CRT-MG and U87-MG, were used, and an agonistic antibody against DR5 (TRA-8) and human recombinant TRAIL were used to ligate DR5. We demonstrate that DR5 ligation by either TRAIL or TRA-8 induces two functional outcomes, apoptosis and expression of the chemokine interleukin-8 (IL-8); the nonspecific caspase inhibitor Boc-D-Fmk blocks both TRAIL-mediated cell death and IL-8 production; the caspase 3-specific inhibitor z-DEVD-Fmk suppresses TRAIL-mediated apoptosis but not IL-8 induction; caspase 1- and 8-specific inhibitors block both TRAIL-mediated cell death and IL-8 production; and DR5 ligation by TRAIL mediates AP-1 and NF-kappaB activation, which can be inhibited by caspase 1- and 8-specific inhibitors. These findings collectively indicate that DR5 ligation on human glioma cells leads to apoptosis and that the activation of AP-1 and NF-kappaB leads to the induction of IL-8 expression; these responses are dependent on caspase activation. Therefore, the TRAIL-DR5 system has a role not only as an inducer of apoptotic cell death but also as a transducer for proinflammatory and angiogenic signals in human brain tumors.     

Cisplatin inactivation of caspases inhibits death ligand-induced cell death in vitro and fulminant liver damage in mice

Cisplatin is a platinum-containing chemotherapeutic drug that has been widely used to treat various human cancers. It acts by forming inter- and intracross-links of DNA, which is believed to be a major cause for its therapeutic efficacy. However, little attention has been paid to the effect of cisplatin on death ligand-induced cell death. Here we demonstrate that cisplatin inhibits death ligand-induced cell death in cell lines in a p53-independent manner. This inhibitory effect of cisplatin on cell death is direct, whereby cisplatin forms a complex with caspases leading to their inactivation. The cisplatin-caspase complex is reversed by the addition of reducing agent dithiothreitol, and caspase activity is regained. In addition, cisplatin shows a death-inhibition effect in in vivo animal models of fulminant liver damage induced by Fas activation and lipopolysaccharide-induced liver shock mediated by tumor necrosis factor-alpha. Together, we demonstrate that cisplatin inhibits cell death induced by death ligands in cell lines and in mice through caspase inactivation.     

CD95 stimulation results in the formation of a novel death effector domain protein-containing complex

Stimulation of CD95 (APO-1/Fas) by its natural ligand CD95L (APO-1L/FasL) leads to the formation of the death-inducing signaling complex. Here we report that upon CD95 stimulation in several T and B cell lines, a novel signaling complex is formed, which we term complex II. Complex II is composed of the death effector domain proteins as follows: procaspase-8a/b, three isoforms of c-FLIP (c-FLIP(L), c-FLIP(S), c-FLIP(R)), and FADD. Notably, complex II does not contain CD95. Based on our findings we suggest that CD95 signaling includes two steps. The first step involves formation of the death-inducing signaling complex at the cell membrane. The second step involves formation of the cytosolic death effector domain protein-containing complex that may play an important role in amplification of caspase activation.     

Necrotic death pathway in Fas receptor signaling

A caspase 8-deficient subline (JB6) of human Jurkat cells can be killed by the oligomerization of Fas-associated protein with death domain (FADD). This cell death process is not accompanied by caspase activation, but by necrotic morphological changes. Here, we show that the death effector domain of FADD is responsible for the FADD-mediated necrotic pathway. This process was accompanied by a loss of mitochondrial transmembrane potential (DeltaPsim), but not by the release of cytochrome c from mitochondria. Pyrrolidine dithiocarbamate, a metal chelator and antioxidant, efficiently inhibited the FADD-induced reduction of DeltaPsim and necrotic cell death. When human Jurkat, or its transformants, expressing mouse Fas were treated with Fas ligand or anti-mouse Fas antibodies, the cells died, showing characteristics of apoptosis. A broad caspase inhibitor (z-VAD-fmk) blocked the apoptotic morphological changes and the release of cytochrome c. However, the cells still died, and this cell death process was accompanied by a strong reduction in DeltaPsim, as well as necrotic morphological changes. The presence of z-VAD-fmk and pyrrolidine dithiocarbamate together blocked cell death, suggesting that both apoptotic and necrotic pathways can be activated through the Fas death receptor.     

Effect of interferon-γ on the susceptibility to Fas (CD95/APO-1)-mediated cell death in human hepatoma cells

Many tumors, including hepatocellular carcinomas (HCCs), resist Fas-mediated cell death, which is one of the effector mechanisms in the host's anti-tumor response; however, this resistance can be abolished by interferon-γ (IFN-γ). IFN-γ may sensitize Fas-mediated cell death in several ways, but the exact mechanism in HCCs is uncertain. In this study, we thoroughly investigated the effect of IFN-γ on the susceptibility of one human normal liver cell line and 12 HCC cell lines to Fas-mediated cell death. We also investigated the effect of IFN-γ on the expression of various apoptosis-related genes such as the Fas/TNF-related genes, the bcl-2 family, and the caspase family of genes. Although most cell lines showed considerable constitutive expression of Fas, all tested cell lines resisted Fas-mediated cell death without IFN-γ. When cells were pretreated with IFN-γ, only three cell lines were made significantly susceptible to Fas-mediated cell death (SNU-354, SNU-387 and SNU-423); the other 10 cell lines were not affected. IFN-γ increased the mRNA expression of Fas, TRAIL and caspase-1, and surface Fas was also increased. The strongly sensitized cell lines (SNU-354, SNU-387 and SNU-423) showed a particularly potent increment in surface Fas after IFN-γ treatment (increase in surface Fas >1.7-fold). This result enabled us to conclude that a potent increment of surface Fas expression is a major sensitizing mechanism of IFN-γ. We conclude that IFN-γ cannot play a sensitizing role in most HCC cell lines and that IFN-γ makes HCC cells susceptible to Fas-mediated cell death through a marked up-regulation of surface Fas in some HCC cells. Received: 3 August 2000 / Accepted: 24 November 2000     

High cell surface death receptor expression determines type I versus type II signaling

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.     

CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily

CD40, a tumor necrosis factor (TNF) receptor (TNFR) family member, conveys signals regulating diverse cellular responses, ranging from proliferation and differentiation to growth suppression and cell death. The ability of CD40 to mediate apoptosis in carcinoma cells is intriguing given the fact that the CD40 cytoplasmic C terminus lacks a death domain homology with the cytotoxic members of the TNFR superfamily, such as Fas, TNFR1, and TNF-related apoptosis-inducing ligand (TRAIL) receptors. In this study, we have probed the mechanism by which CD40 transduces death signals. Using a trimeric recombinant soluble CD40 ligand to activate CD40, we have found that this phenomenon critically depends on the membrane proximal domain (amino acids 216 to 239) but not the TNFR-associated factor-interacting PXQXT motif in the CD40 cytoplasmic tail. CD40-mediated cytotoxicity is blocked by caspase inhibitors, such as zVAD-fmk and crmA, and involves activation of caspase 8 and caspase 3. Interestingly, CD40 ligation was found to induce functional Fas ligand, TRAIL (Apo-2L) and TNF in apoptosis-susceptible carcinoma cells and to up-regulate expression of Fas. These findings identify a novel proapoptotic mechanism which is induced by CD40 in carcinoma cells and depends on the endogenous production of cytotoxic cytokines and autocrine or paracrine induction of cell death.     

Mitochondrial regulation of cell death: mitochondria are essential for procaspase 3-p21 complex formation to resist Fas-mediated cell death

Death receptor Fas transduces cell death signaling upon stimulation by Fas ligand, and this death signaling is mediated by caspase. Recently, we reported that the cell cycle regulator p21 interacts with procaspase 3 to resist Fas-mediated cell death. In the present study, the molecular characterization and functional region of the procaspase 3-p21 complex was further investigated. We observed the p21 expression in the mitochondrial fraction of HepG2 cells and detected Fas-mediated cell death only in the presence of actinomycin D. However, mitochondrial-DNA-lacking HepG2 (MDLH) cells showed this effect even in the absence of actinomycin D. Both p21 and procaspase 3 were expressed in MDLH cells, but the procaspase 3-p21 complex formation was not observed. Interestingly, the resistance to Fas-mediated cell death in the MDLH cells without actinomycin D was recovered after microinjection of HepG2-derived mitochondria into the MDLH cells. We conclude that mitochondria are necessary for procaspase 3-p21 complex formation and propose that the mitochondrial role during cell death is not only death induction but also death suppression.     

Rapid induction of apoptosis in CD8+ HIV-1 envelope-specific murine CTLs by short exposure to antigenic peptide

During primary viral infection, in vivo exposure to high doses of virus causes a loss of Ag-specific CD8(+) T cells. This phenomenon, termed clonal exhaustion, and other mechanisms by which CTLs are deleted are poorly understood. Here we show evidence for a novel form of cell death in which recently stimulated CD8(+) HIV-1 envelope gp160-specific murine CTLs become apoptotic in vitro after brief exposure to free antigenic peptide (P18-I10). Peak apoptosis occurred within 3 h of treatment with peptide, and the level of apoptosis was dependent on both the time after initial stimulation with target cells and the number of targets. Using T cell-specific H-2D(d)/P18-I10 tetramers, we observed that the apoptosis was induced by such complexes. Induction of apoptosis was blocked by cyclosporin A, a caspase 3 inhibitor, and a mitogen-activated protein kinase inhibitor, but not by Abs to either Fas ligand or to TNF-alpha. Thus, these observations suggest the existence of a Fas- or TNF-alpha-independent pathway initiated by TCR signaling that is involved in the rapid induction of CTL apoptosis. Such a pathway may prove important in the mechanism by which virus-specific CTLs are deleted in the presence of high viral burdens.     

Different programs of activation-induced cell death are triggered in mature activated CTL by immunogenic and partially agonistic peptide ligands

Activation-induced cell death (AICD) of mature T cells plays an important role in the control of immune homeostasis and peripheral tolerance. TNFRs and Fas have been implicated in the induction of AICD. However, these molecules were shown to be dispensable, at least in some experimental systems, for downsizing of Ag-induced T cell expansions and development of tolerance in vivo. The conditions of T cell activation leading to T cell deletion in a death receptor-independent manner are not well characterized. Here we show that human CTLs die through a death receptor-independent apoptotic program upon triggering with a partially agonistic peptide ligand. This apoptotic process exhibits some features of T cell death due to lymphokine deprivation and is blocked by exogenous IL-2. Our data demonstrate that engagement of TCR by MHC-peptide complexes can trigger diverse apoptotic programs of AICD and that the choice between these programs is determined by the agonistic potency of MHC-peptide ligand.