MEK1 activation rescues Jurkat T cells from Fas-induced apoptosis.

Although the protease cascade initiated by Fas (CD95, Apo-1) is well characterized, there remains little known about how kinase pathways may impact on Fas-mediated apoptosis. We recently observed that in T lymphocytes Fas strongly induced activation of JNK (c-Jun N-terminal kinase) but not of second messengers leading to activation of ERK (extracellular regulated kinase). Additionally, Fas-mediated apoptosis was significantly inhibited with PMA, a potent activator of the ERK signaling pathway. This suggested a model whereby activation of the ERK pathway might attenuate Fas-mediated apoptosis. This was confirmed in the current study by showing that activation of MEK1, the upstream regulator of ERK, reduces Fas-mediated apoptosis, whereas inhibition of MEK1 augments apoptosis by Fas. Furthermore, Fas-mediated apoptosis of Jurkat T cells is not affected by constitutively active or dominant negative variants that modulate the JNK pathway. These results demonstrate that Fas-induced JNK activation is not required for apoptosis by Jurkat T cells, but rather is more likely secondary to cell stress during the early phases of apoptosis. This is supported by the ability of the caspase blocker zVAD to inhibit both apoptosis and JNK activation by Fas.     

Epidermal growth factor protects epithelial cells against Fas-induced apoptosis. Requirement for Akt activation.

Chemotherapeutic drugs that damage DNA kill tumor cells, in part, by inducing the expression of a death receptor such as Fas or its ligand, FasL. Here, we demonstrate that epidermal growth factor (EGF) stimulation of T47D breast adenocarcinoma and embryonic kidney epithelial (HEK293) cells protects these cells from Fas-induced apoptosis. EGF stimulation of epithelial cells also inhibited Fas-induced caspase activation and the proteolysis of signaling proteins downstream of the EGF receptor, Cbl and Akt/protein kinase B (Akt). EGF stimulation of Akt kinase activity blocked Fas-induced apoptosis. Expression of activated Akt in MCF-7 breast adenocarcinoma cells was sufficient to block Fas-mediated apoptosis. Inhibition of EGF-stimulated extracellular signal-regulated kinase (ERK) activity did not affect EGF protection from Fas-mediated apoptosis. The findings indicate that EGF receptor stimulation of epithelial cells has a significant survival function against death receptor-induced apoptosis mediated by Akt.     

p38 mitogen-activated protein kinase mediates the Fas-induced mitochondrial death pathway in CD8+ T cells

The p38 mitogen-activated protein kinase (MAPK) signaling pathway can be activated by a variety of stress stimuli such as UV radiation and osmotic stress. The regulation and role of this pathway in death receptor-induced apoptosis remain unclear and may depend on the specific death receptor and cell type. Here we show that binding of Fas ligand to Fas activates p38 MAPK in CD8+ T cells and that activation of this pathway is required for Fas-mediated CD8+ T-cell death. Active p38 MAPK phosphorylates Bcl-xL and Bcl-2 and prevents the accumulation of these antiapoptotic molecules within the mitochondria. Consequently, a loss of mitochondrial membrane potential and the release of cytochrome c lead to the activation of caspase 9 and, subsequently, caspase 3. Therefore, the activation of p38 MAPK is a critical link between Fas and the mitochondrial death pathway and is required for the Fas-induced apoptosis of CD8+ T cells.     

FADD is required for multiple signaling events downstream of the receptor Fas.

To identify essential components of the Fas-induced apoptotic signaling pathway, Jurkat T lymphocytes were chemically mutagenized and selected for clones that were resistant to Fas-induced apoptosis. We obtained five cell lines that contain mutations in the adaptor FADD. All five cell lines did not express FADD by immunoblot analysis and were completely resistant to Fas-induced death. Complementation of the FADD mutant cell lines with wild-type FADD restored Fas-mediated apoptosis. Fas activation of caspase-2, caspase-3, caspase-7, and caspase-8 and the proteolytic cleavage of substrates such as BID, protein kinase Cdelta, and poly(ADP-ribose) polymerase were completely defective in the FADD mutant cell lines. In addition, Fas activation of the stress kinases p38 and c-Jun NH2 kinase and the generation of ceramide in response to Fas ligation were blocked in the FADD mutant cell lines. These data indicate that FADD is essential for multiple signaling events downstream of Fas.     

Oxidative stress-induced apoptosis in retinal photoreceptor cells is mediated by calpains and caspases and blocked by the oxygen radical scavenger CR-6

A critical role for reactive oxygen species (ROS) in photoreceptor apoptosis has been established. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. This study delineates the molecular events that occur after treatment of the photoreceptor cell line 661W with the nitric oxide donor sodium nitroprusside (SNP). Cytosolic calcium levels increased during photoreceptor apoptosis, leading to activation of the calcium-dependent proteases calpains. Furthermore, caspase activation also occurred following SNP insult. However, although treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone inhibited caspase activity per se in SNP-treated 661W cells, it did not prevent apoptosis. On the other hand, CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acted as a scavenger of ROS and reduced 661W photoreceptor apoptosis induced by SNP by preventing the activation of a pathway in which calpains have a key role. In summary, we report for the first time that both caspases and calpains are involved in 661W photoreceptor apoptosis and that calpain activation can be prevented by the ROS scavenger CR-6.     

Fas aggregation does not correlate with Fas-mediated apoptosis.

Cross-linking of cell surface Fas molecules by Fas ligand or by agonistic anti-Fas Abs induces cell death by apoptosis. We found that a serine protease inhibitor, N-tosyl-L-lysine chloromethyl ketone (TLCK), dramatically enhances Fas-mediated apoptosis in the human T cell line Jurkat and in various B cell lines resistant to Fas-mediated apoptosis. The enhancing effect of TLCK is specific to Fas-induced cell death, with no effect seen on TNF-alpha or TNF-related apoptosis-inducing ligand-induced apoptosis. TLCK treatment had no effect on Fas expression levels on the cell surface, and neither promoted death-inducing signaling complex formation nor decreased expression levels of cellular inhibitors of apoptosis (FLICE inhibitory protein, X chromosome-linked inhibitor of apoptosis, and Bcl-2). Activation of the Fas-mediated apoptotic pathway by anti-Fas Ab is accompanied by aggregation of Fas molecules to form oligomers that are stable to boiling in SDS and beta-ME. Fas aggregation is often considered to be required for Fas-mediated apoptosis. However, sensitization of cells to Fas-mediated apoptosis by TLCK or other agents (cycloheximide, protein kinase C inhibitors) causes less Fas aggregation during the apoptotic process compared with that in nonsensitized cells. These results show that Fas aggregation and Fas-mediated apoptosis are not directly correlated and may even be inversely correlated.     

Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis

Resistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2-related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)-induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system.     

Cell cycle-dependent regulation of FLIP levels and susceptibility to Fas-mediated apoptosis

Activation-induced cell death of peripheral T cells results from the interaction between Fas and Fas ligand. Resting peripheral T cells are resistant to Fas-induced apoptosis and become susceptible only after their activation. We have investigated the molecular mechanism mediating the sensitization of resting peripheral T cells to Fas-mediated apoptosis following TCR stimulation. TCR activation decreases the steady state protein levels of FLIP (FLICE-like inhibitory protein), an inhibitor of the Fas signaling pathway. Reconstitution of intracellular FLIP levels by the addition of a soluble HIV transactivator protein-FLIP chimera completely restores resistance to Fas-mediated apoptosis in TCR primary T cells. Inhibition of IL-2 production by cyclosporin A, or inhibition of IL-2 signaling by rapamycin or anti-IL-2 neutralizing Abs prevents the decrease in FLIP levels and confers resistance to Fas-mediated apoptosis following T cell activation. Using cell cycle-blocking agents, we demonstrate that activated T cells arrested in G1 phase contain high levels of FLIP protein, whereas activated T cells arrested in S phase have decreased FLIP protein levels. These findings link regulation of FLIP protein levels with cell cycle progression and provide an explanation for the increase in TCR-induced apoptosis observed during the S phase of the cell cycle.     

Ceramide is the key mediator of oxidative stress-induced apoptosis in retinal photoreceptor cells

Nitric oxide and reactive oxygen species play a critical role in photoreceptor apoptosis. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. Here, we demonstrate that the sphingolipid ceramide is the key mediator of oxidative stress-induced apoptosis in 661W retinal photoreceptor cells. Treatment of 661W cells with the nitric oxide donor, sodium nitroprusside, activates acid sphingomyelinase. As a result, sphingomyelin is hydrolysed, which leads to an increase in the concentration of ceramide. We also show that ceramide is responsible for the activation of the mitochondrial apoptotic pathway in 661W photoreceptor cells and subsequent activation of the caspase cascade. Furthermore, we show for the first time that ceramide is responsible for the increased Ca2+ levels in the mitochondria and cytosol that precedes activation of the calpain-mediated apoptotic pathway. Additionally, we provide evidence that ceramide also activates the endolysosomal protease cathepsin D pathway. In summary, our findings show that ceramide controls the cell death decisions in photoreceptor cells and highlight the relevance of acid sphingomyelinase as a potential therapeutic target for the treatment of retinal pathologies.     

Induction and regulation of Fas-mediated apoptosis in human thyroid epithelial cells

Fas-mediated apoptosis has been proposed to play an important role in the pathogenesis of Hashimoto's thyroiditis. Normal thyroid cells are resistant to Fas-mediated apoptosis in vitro but can be sensitized by the unique combination of interferon-gamma and IL-1beta cytokines. We sought to examine the mechanism of this sensitization and apoptosis signaling in primary human thyroid cells. Without the addition of cytokines, agonist anti-Fas antibody treatment of the thyroid cells resulted in the cleavage of proximal caspases, but this did not lead to the activation of caspase 7 and caspase 3. Apoptosis associated with the cleavage of caspases 7, 3, and Bid, and the activation of mitochondria in response to anti-Fas antibody occurred only after cytokine pretreatment. Cell surface expression of Fas, the cytoplasmic concentrations of procaspases 7, 8, and 10, and the proapoptotic molecule Bid were markedly enhanced by the presence of the cytokines. In contrast, P44/p42 MAPK (Erk) appeared to provide protection from Fas-mediated apoptosis because an MAPK kinase inhibitor (U0126) sensitized thyroid cells to anti-Fas antibody. In conclusion, Fas signaling is blocked in normal thyroid cells at a point after the activation of proximal caspases. Interferon-gamma/IL-1beta pretreatment sensitizes human thyroid cells to Fas-mediated apoptosis in a complex manner that overcomes this blockade through increased expression of cell surface Fas receptor, increases in proapoptotic molecules that result in mitochondrial activation, and late caspase cleavage. This process involves Bcl-2 family proteins and appears to be compatible with type II apoptosis regulation.     

Calmodulin mediates Fas-induced FADD-independent survival signaling in pancreatic cancer cells via activation of Src-extracellular signal-regulated kinase (ERK)

Pancreatic cancer remains a devastating malignancy with a poor prognosis and is largely resistant to current therapies. To understand the resistance of pancreatic tumors to Fas death receptor-induced apoptosis, we investigated the molecular mechanisms of Fas-activated survival signaling in pancreatic cancer cells. We found that knockdown of the Fas-associated protein with death domain (FADD), the adaptor that mediates downstream signaling upon Fas activation, rendered Fas-sensitive MiaPaCa-2 and BxPC-3 pancreatic cells resistant to Fas-induced apoptosis. By contrast, Fas activation promoted the survival of the FADD knockdown MiaPaCa-2 and BxPC-3 cells in a concentration-dependent manner. The pharmacological inhibitor of ERK, PD98059, abrogated Fas-promoted cell survival in FADD knockdown MiaPaCa-2 and BxPC-3 cells. Furthermore, increased phosphorylation of Src was demonstrated to mediate Fas-induced ERK activation and cell survival. Immunoprecipitation of Fas in the FADD knockdown cells identified the presence of increased calmodulin, Src, and phosphorylated Src in the Fas-associated protein complex upon Fas activation. Trifluoperazine, a calmodulin antagonist, inhibited Fas-induced recruitment of calmodulin, Src, and phosphorylated Src. Consistently, trifluoperazine blocked Fas-promoted cell survival. A direct interaction of calmodulin and Src and their binding site were identified with recombinant proteins. These results support an essential role of calmodulin in mediating Fas-induced FADD-independent activation of Src-ERK signaling pathways, which promote survival signaling in pancreatic cancer cells. Understanding the molecular mechanisms responsible for the resistance of pancreatic cells to apoptosis induced by Fas-death receptor signaling may provide molecular insights into designing novel therapies to treat pancreatic tumors.     

Reactive oxygen species are involved in FasL-induced caspase-independent cell death and inflammatory responses

Fas-mediated caspase-dependent cell apoptosis has been well investigated. However, recent studies have shown that Fas can induce nonapoptotic caspase-independent cell death (CICD) when caspase activity is inhibited. Currently, the molecular mechanism of this alternative cell death mediated by Fas remains unclear. In this study, we investigated the signaling pathway of Fas-induced CICD in mouse embryonic fibroblasts (MEFs) whose caspase function was disrupted by the pan-caspase inhibitor Z-VAD-FMK and its coupling to inflammatory responses. Our results revealed that receptor-interacting protein 1 and tumor necrosis factor receptor-associated factor 2 play important roles in FasL-induced CICD. This death is associated with intracellular reactive oxygen species (ROS) production from mitochondria, as a ROS scavenger (BHA), antioxidants (trolox, NAC), and a mitochondrial respiratory chain uncoupler (rotenone) could prevent this event. Furthermore, delayed and sustained JNK activation, mitochondrial membrane potential breakdown, and loss of intracellular GSH were observed. In addition to CICD, FasL also induces cyclooxygenase-2 and MIP-2 gene upregulation, and both responses are attributed to ROS-dependent JNK activation. Taken together, these results demonstrate alternative signaling pathways of Fas upon caspase inhibition in MEFs that are unrelated to the classical apoptotic pathway, but steer cells toward necrosis and an inflammatory response through ROS production.     

Inflammatory cytokine regulation of Fas-mediated apoptosis in thyroid follicular cells.

The occurrence of apoptosis in thyroid follicular cells induced by Fas activation has been a subject of much debate. This is due, in part, to the fact that no physiologically relevant treatment conditions have been reported to cause rapid and extensive Fas-mediated apoptosis in thyroid cells, whereas treatment with the protein synthesis inhibitor cycloheximide prior to Fas activation allows for massive cell death. This indicates that the Fas signaling pathway is present but that its function is blocked in the overwhelming majority of cultured thyroid cells. To reconcile the conflicting reports, we set out to identify physiologically relevant conditions in which rapid, massive thyroid cell apoptosis in response to Fas activation could be demonstrated. We determined that susceptibility to Fas-activated apoptosis could be influenced by certain combinations of inflammatory cytokines. Although no single cytokine was effective, pretreatment of thyroid cells with the combination of gamma-interferon and either tumor necrosis factor-alpha or interleukin 1beta allowed for massive Fas-mediated apoptosis. Susceptibility to Fas-induced death correlated with an increase in expression of a tunicamycin-inhibitable high molecular weight form of Fas but not with aggregate expression of Fas.     

Calmodulin binding to cellular FLICE-like inhibitory protein modulates Fas-induced signalling

We and others have demonstrated that Fas-mediated apoptosis is a potential therapeutic target for cholangiocarcinoma. Previously, we reported that CaM (calmodulin) antagonists induced apoptosis in cholangiocarcinoma cells through Fas-related mechanisms. Further, we identified a direct interaction between CaM and Fas with recruitment of CaM into the Fas-mediated DISC (death-inducing signalling complex), suggesting a novel role for CaM in Fas signalling. Therefore we characterized the interaction of CaM with proteins recruited into the Fas-mediated DISC, including FADD (Fas-associated death domain)-containing protein, caspase 8 and c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like interleukin 1beta-converting enzyme]-like inhibitory protein}. A Ca(2+)-dependent direct interaction between CaM and FLIP(L), but not FADD or caspase 8, was demonstrated. Furthermore, a 37.3+/-5.7% increase (n=6, P=0.001) in CaM-FLIP binding was observed at 30 min after Fas stimulation, which returned to the baseline after 60 min and correlated with a Fas-induced increase in intracellular Ca(2+) that reached a peak at 30 min and decreased gradually over 60 min in cholangiocarcinoma cells. A CaM antagonist, TFP (trifluoperazine), inhibited the Fas-induced increase in CaM-FLIP binding concurrent with inhibition of ERK (extracellular-signal-regulated kinase) phosphorylation, a downstream signal of FLIP. Direct binding between CaM and FLIP(L) was demonstrated using recombinant proteins, and a CaM-binding region was identified in amino acids 197-213 of FLIP(L). Compared with overexpression of wild-type FLIP(L) that resulted in decreased spontaneous as well as Fas-induced apoptosis, mutant FLIP(L) with deletion of the CaM-binding region resulted in increased spontaneous and Fas-induced apoptosis in cholangiocarcinoma cells. Understanding the biology of CaM-FLIP binding may provide new therapeutic targets for cholangiocarcinoma and possibly other cancers.     

Activation of the Rb/E2F1 pathway by the nonproliferative p38 MAPK during Fas (APO1/CD95)-mediated neuronal apoptosis

Aberrant activation of the Rb/E2F1 pathway in cycling cells, in response to mitogenic or nonmitogenic stress signals, leads to apoptosis through hyperphosphorylation of Rb. To test whether in postmitotic neurons the Rb/E2F1 pathway can be activated by the nonmitogenic stress signaling, we examined the role of the p38 stress-activated protein kinase (SAPK) in regulating Rb phosphorylation in response to Fas (CD95/APO1)-mediated apoptosis of cultured cerebellar granule neurons (CGNs). Anti-Fas antibody induced a dramatic and early activation of p38. Activated p38 was correlated with the induction of hyperphosphorylation of both endogenous and exogenous Rb. The p38-selective inhibitor, SB203580, attenuated such an increase in pRb phosphorylation and significantly protected CGNs from Fas-induced apoptosis. The cyclin-dependent kinase-mediated Rb phosphorylation played a lesser role in this neuronal death paradigm, since cyclin-dependent kinase inhibitors, such as olomoucine, roscovitine, and flavopiridol, did not significantly prevent anti-Fas antibody-evoked neuronal apoptosis. Hyperphosphorylation of Rb by p38 SAPK resulted in the release of Rb-bound E2F1. Increased E2F1 modulated neuronal apoptosis, since E2F1-/- CGNs were significantly less susceptible to Fas-mediated apoptosis in comparison with the wild-type CGNs. Taken together, these studies demonstrate that neuronal Rb/E2F1 is modulated by the nonproliferative p38 SAPK in Fas-mediated neuronal apoptosis.     

Resistance to Fas-mediated apoptosis in EBV-infected B cell lymphomas is due to defects in the proximal Fas signaling pathway.

Post-transplant lymphoproliferative disorder is characterized by the outgrowth of EBV-infected B cell lymphomas in immunosuppressed transplant recipients. Using a panel of EBV-infected spontaneous lymphoblastoid cell lines (SLCL) derived from post-transplant lymphoproliferative disorder patients, we assessed the sensitivity of such lymphomas to Fas-mediated cell death. Treatment with either an agonist anti-Fas mAb or Fas ligand-expressing cells identifies two subsets of SLCL based on their sensitivity or resistance to Fas-driven apoptosis. Fas resistance in these cells cannot be attributed to reduced Fas expression or to mutations in the Fas molecule itself. In addition, all SLCL are sensitive to staurosporine-induced cell death, indicating that there is no global defect in apoptosis. Although all SLCL express comparable levels of Fas signaling molecules including Fas-associated death domain protein, caspase 8, and caspase 3, Fas-resistant SLCL exhibit a block in Fas-signaling before caspase 3 activation. In two SLCL, this block results in impaired assembly of the death-inducing signaling complex, resulting in reduced caspase 8 activation. In a third Fas-resistant SLCL, caspase 3 activation is hindered despite intact death-inducing signaling complex formation and caspase 8 activation. Whereas multiple mechanisms exist by which tumor cells can evade Fas-mediated apoptosis, these studies suggest that the proximal Fas-signaling pathway is impeded in Fas-resistant post-transplant lymphoproliferative disorder-associated EBV(+) B cell lymphomas.     

Lyme arthritis synovial gammadelta T cells instruct dendritic cells via fas ligand

gammadelta T cells participate in the innate immune response to a variety of infectious microorganisms. They also link to the adaptive immune response through their induction of maturation of dendritic cells (DC) during the early phase of an immune response when the frequency of Ag-specific T cells is very low. We observe that in the presence of Borrelia burgdorferi, synovial Vdelta1 T cells from Lyme arthritis synovial fluid potently induce maturation of DC, including production of IL-12, and increased surface expression of CD40 and CD86. The activated DC are then able to stimulate the Vdelta1 T cells to up-regulate CD25. Both of these processes are initiated primarily by Fas stimulation rather than CD40 activation of DC via high expression of Fas ligand by the Vdelta1 T cells. DC are resistant to Fas-induced death due to expression of high levels of the Fas inhibitor c-FLIP. This effect serves to divert Fas-mediated signals from the caspase cascade to the ERK MAPK and NF-kappaB pathways. The findings affirm the importance of the interaction of certain T cell populations with DC during the early phases of the innate immune response. They also underscore the view that as levels of c-FLIP increase, Fas signaling can be diverted from induction of apoptosis to pathways leading to cell effector function.     

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.     

Distinct molecular mechanisms of Fas resistance in murine B lymphoma cells

A panel of murine B lymphoma cell lines, which express different levels of Fas, was extensively studied for sensitivity to Fas-mediated death signals via an anti-Fas mAb and Fas ligand-bearing cell lines. Expression of the Fas receptor on the B lymphoma cell lines did not correlate with their capacity to undergo Fas-mediated apoptosis. Moreover, Fas-associated death domain protein recruitment to the death-inducing signaling complex (DISC) complex occurred in all cell lines expressing Fas, regardless of whether they were sensitive to Fas-mediated death. Interestingly, the protein synthesis inhibitor, cycloheximide, and protein kinase C inhibitors, such as bisindolylmaleimide, rendered one of the resistant cell lines, CH33, sensitive to signals from the Fas receptor, although the levels of Fas were unchanged. This suggests that constitutive PKC activation plays a role in Fas resistance, perhaps by up-regulating NF-kappaB or Bcl-2 family members. Interestingly, CH33 demonstrated caspase 8 activity upon engagement of the Fas receptor in the absence of pharmacological manipulation, suggesting that the block in apoptosis is downstream of the DISC complex. In contrast, the fact that Fas-associated death domain protein was recruited to the DISC complex in other resistant lines, such as WEHI-231, with no caspase 8 activation indicates that these cells may be blocked within the DISC complex. Indeed, Western blot analysis showed that WEHI-231 expressed an isoform of FLICE-like inhibitory protein (cFLIPL), an antiapoptotic protein within the DISC. These studies provide evidence that murine B lymphoma cells utilize different molecular mechanisms along the Fas-signaling cascade to block apoptosis.