Characterization of apoptosis pathways (intrinsic and extrinsic) in lymphoid tissues of calves inoculated with non-cytopathic bovine viral diarrhoea virus genotype-1

Previous studies have shown that activation of effector caspase-3 is associated with the apoptosis of lymphocytes occurring during infection with bovine viral diarrhoea virus (BVDV); however, the regulation of the apoptosis pathways that induce cell death via activation of effector caspase-3 has not yet been clarified. The aim of this study was to examine immunohistochemically the expression of cleaved caspase (CCasp)-8 (initiator caspase of the extrinsic pathway), CCasp9 (initiator caspase of the intrinsic pathway) and Bcl-2 (an anti-apoptotic marker) in gut-associated lymphoid tissue (GALT) of the ileum from calves inoculated with a non-cytopathic strain of BVDV genotype-1. CCasp8 had similar expression to that of CCasp3. In interfollicular T-cell areas there was moderate apoptosis and evidence of moderate activation of initiator caspase-8. In B-cell follicles there was marked lymphocyte apoptosis and evidence of intense caspase-8 activation, highlighting the potentially major role of the extrinsic pathway in lymphocyte apoptosis in the GALT during BVDV infection. Additionally, there was a significant decrease in the number of CCasp9(+) cells from the start of the experiment and this was linked to inactivation of caspase-9. Therefore, the intrinsic pathway may play only a minor role in the induction of lymphocyte apoptosis. Finally, the observed overexpression of Bcl-2 protein could play a major role in protecting lymphocytes in the T-cell areas against apoptosis, while low levels of Bcl-2 expression could be associated with the follicular lymphocyte apoptosis occurring during BVDV infection.     

Stomach cancer highly expresses both initiator and effector caspases; an immunohistochemical study

Caspases play an essential role during apoptotic cell death. While caspases 8 and 10 act as initiator caspases of the extrinsic apoptosis pathway, caspase 9 acts as an initiator caspase of the intrinsic apoptosis pathway. Caspase 3 is considered to be the main effector caspase involved in both intrinsic and extrinsic pathways. Alteration of apoptosis is essential for cancer development. Thus, analysis of the expression status of caspases, the main executioners of apoptosis, in cancer tissues is needed for a sophisticated understanding of cancer biology. In the current study, we analyzed the expression of caspases 3, 8, 9 and 10 in 60 advanced gastric adenocarcinomas by immunohistochemistry using a tissue microarray approach. Immunopositivity was observed for caspase 3 in 57 (95%), caspase 8 in 56 (93%), caspase 9 in 54 (90%), and caspase 10 in 58 (97%) of the 60 cancers. While 46 cancers (77%) expressed all of the caspases examined, 14 cancers (23%) showed loss of expression in one or more caspases examined. Normal gastric mucosal cells showed no or weak expression of caspases 3, 8, 9 and 10. Taken together, these results suggest that stomach cancer cells in vivo may need caspase expression for apoptosis. Also, higher expression of the caspases in stomach cancer cells than in normal gastric mucosal cells suggests that apoptosis in susceptible stomach cancer cells might be easily triggered, thereby producing selective pressure to make more apoptosis-resistant cells during tumor development.     

Fas/CD95-mediated apoptosis of type II cells is blocked by Toxoplasma gondii primarily via interference with the mitochondrial amplification loop

The intracellular protozoan Toxoplasma gondii induces persistent infections in various hosts and is an important opportunistic pathogen of humans with immature or deficient immune responses. The ability to survive intracellularly largely depends on the blocking of different proapoptotic signaling cascades of its host cell. Fas/CD95 triggers an apoptotic cascade that is crucial for immunity and the outcome of infectious diseases. We have determined the mechanism by which T. gondii counteracts death receptor-mediated cell death in type II cells that transduce Fas/CD95 ligation via caspase 8-mediated activation of the mitochondrial amplification loop. The results showed that infection with T. gondii significantly reduced Fas/CD95-triggered apoptosis in HeLa cells by inhibiting the activities of initiator caspases 8 and 9 and effector caspase 3/7. Parasitic infection dose dependently diminished cleavage of caspase 8, the BH3-only protein Bid, and the downstream caspases 9 and 3. Importantly, interference with Fas/CD95-triggered caspase 8 and caspase 3/7 activities after parasitic infection was largely dependent on the presence of caspase 9. Within the mitochondrial amplification loop, T. gondii significantly inhibited the Fas/CD95-triggered release of cytochrome c into the host cell cytosol. These results indicate that T. gondii inhibits Fas/CD95-mediated apoptosis in type II cells primarily by decreasing the apoptogenic function of mitochondria.     

Interaction with XIAP prevents full caspase-3/-7 activation in proliferating human T lymphocytes

Caspases are essential mediators of cytokine release and apoptosis. Additionally, caspase activity is required for the proliferation of naive T lymphocytes. It remained unclear how proliferating cells are able to cope with the pro-apoptotic activity especially of effector caspases-3 and -7. Possible reasons might include limited subcellular localization of active caspases or inhibition by endogenous caspase inhibitors. Here, we compared the activation of various caspases in proliferating human T cells with that in apoptotic cells. We show that cleaved caspases-3/-7 appear to be widely distributed in apoptotic cells while they are largely confined to the cytoplasm in proliferating cells. Additionally, in proliferating T cells caspase-3 remains incompletely cleaved, while in apoptotic cells fully mature caspase-3 is generated. We provide evidence that during T cell proliferation the intracellular caspase inhibitor X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspases-3/-7, thereby blocking their full activation, substrate cleavage, and cell death. The lack of substrate cleavage might also lead to the observed limited subcellular distribution of caspases-3/-7. After induction of apoptosis, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point (Smac/DIABLO) is released from mitochondria, resulting in the abrogation of the inhibitory effect of XIAP, full activation of caspases-3/-7, and apoptosis.     

Genome-wide screening and identification of novel proteolytic cleavage targets of caspase-8 and -10 in vitro

Apoptosis executed by the mammalian caspase family plays a fundamental role in cellular homeostasis. Deregulation of this process is associated with several human diseases. The multimerization of ligand-induced death receptors results in the recruitment of the death inducing signaling complex and autocatalytic activation of initiator caspases, including caspase-8 and -10. However, it is still unclear how initiator caspases trigger and control the early apoptotic signaling pathways, partly because the downstream proteolytic cleavage targets of the initiator caspases are not completely known. Although it is known that a number of proteins are cleaved by various members of the caspase family, the identification of specific cleavage substrates of the initiator caspases 8 and 10, has been hindered by a lack of systematic and broadly applicable strategies for substrate identification. In the present study we constructed a mouse cDNA library and used it to perform a systematic, genome-wide screen for novel in vitro substrates of caspase-8 and -10. From this, we successfully identified six putative caspase substrates, including five novel proteins (ABCF1, AKAP1, CPE, DOPEY1 and GOPC1) that may be targeted specifically by the initiator caspases 8 and 10 during the early stages of apoptosis. These findings may provide useful information for elucidating the apoptotic signaling pathways downstream of the death receptors.     

Cleavage of FLICE (caspase-8) by granzyme B during cytotoxic T lymphocyte-induced apoptosis

Cytotoxic T lymphocytes induce apoptosis in target cells through the CD95(APO-1/Fas) and the perforin/granzyme B (GrB) pathway. The exact substrate of GrB in vivo is still unknown, but to induce apoptosis GrB requires the activity of caspases in target cells. We show here that in HeLa target cells induction of apoptosis through the perforin/GrB pathway resulted in minor direct cleavage of CPP32 (caspase-3) by GrB. Most caspase-3 cleavage resulted from activation of an upstream caspase. Moreover, target cells derived from caspase-3^?/? mice displayed GrB-induced poly(ADP-ribose) polymerase (PARP) cleavage with only partially reduced efficiency compared to wild-type target cells. This indicates that other PARP-cleaving caspases can be activated during perforin/GrB-induced cell death. In contrast to caspase-3, FLICE (caspase-8) was directly cleaved by GrB in HeLa cells. We therefore conclude that FLICE not only plays a central role in CD95(APO-1/Fas)-induced apoptosis but can also be directly activated during perforin/GrB-induced apoptosis.     

Porcine reproductive and respiratory syndrome virus induces apoptosis through a mitochondria-mediated pathway

As with a number of other viruses, Porcine reproductive and respiratory syndrome virus (PRRSV) has been shown to induce apoptosis, although the mechanism(s) involved remain unknown. In this study we have characterized the apoptotic pathways activated by PRRSV infection. PRRSV-infected cells showed evidence of apoptosis including phosphatidylserine exposure, chromatin condensation, DNA fragmentation, caspase activation (including caspase-8, 9, 3), and PARP cleavage. DNA fragmentation was dependent on caspase activation but blocking apoptosis by a caspase inhibitor did not affect PRRSV replication. Upregulation of Bax expression by PRRSV infection was followed by disruption of the mitochondria transmembrane potential, resulting in cytochrome c redistridution to the cytoplasm and subsequent caspase-9 activation. A crosstalk between the extrinsic and intrinsic pathways was demonstrated by dependency of caspase-9 activation on active caspase-8 and by Bid cleavage. Furthermore, in this study we provide evidence of the possible involvement of reactive oxygen species (ROS)-mediated oxidative stress in apoptosis induced by PRRSV. Our data indicated that cell death caused by PRRSV infection involves necrosis as well as apoptosis. In summary, these findings demonstrate mechanisms by which PRRSV induces apoptosis and will contribute to an enhanced understanding of PRRSV pathogenesis.     

Proliferating γδ T cells manifest high and spatially confined caspase-3 activity

Caspase-8 serves two paradoxical roles in T lymphocytes: it initiates apoptosis following death receptor engagement, and is also indispensible for proliferation following T-cell antigen receptor (TCR) signalling. These opposing processes appear to be controlled by both spatial and quantitative differences in caspase-8 activation. Given differences in the turnover of T-cell subsets, we compared caspase activity and susceptibility to cell death following TCR restimulation in murine CD4(+) and CD8(+) αβ T cells and γδ T cells. We observed a spectrum of caspase activity in non-dying effector T cells in which CD4(+) T cells manifested the lowest levels of active caspases whereas γδ T cells manifested the highest levels. Further analysis revealed that most of the difference in T-cell subsets was the result of high levels of active caspase-3 in non-dying effector γδ T cells. Despite this, γδ T cells manifested little spontaneous or CD3 restimulation-induced cell death as the result of confinement of active caspases to the cell membrane. By contrast, CD4(+) T cells were highly sensitive to CD3-induced cell death, associated with the appearance of active caspases in the cytoplasm and cleavage of the caspase substrates Bid and ICAD. Hence, the location and amount of active caspases distinguishes effector T-cell subsets and profoundly influences the fate of the T-cell response.     

Autoantibodies from Sjögren's syndrome induce activation of both the intrinsic and extrinsic apoptotic pathways in human salivary gland cell line A-253

Sj?gren's syndrome (SS) is an autoimmune rheumatic disease that targets salivary and lachrymal glands, characterized by a high concentration of serum autoantibodies directed against nuclear and cytoplasmic antigens. It is known that autoantibodies can enter viable cells and this phenomenon has functional consequences including activation of apoptotic process. The objective of this work was to explore whether autoantibodies contained in IgG purified from Sj?gren sera trigger apoptotic process in an experimental model represented by the human salivary gland cell line A-253. To define if the intrinsic or extrinsic pathways are activated, we examined which caspases are critical for inducing cell death. The results have demonstrated that morphological changes and DNA laddering, consistent with apoptotic cell death, occurred in A-253 cells treated with IgG from Sj?gren sera. Sj?gren IgG induced cleavage and activation of the effector caspase-3 and degradation of the caspase-3 substrate poly(ADP-ribose)polymerase. Both the intrinsic and extrinsic apoptotic pathways were activated, since both caspase-8 and caspase-9 cleavages occurred. In conclusion, autoantibodies contained in IgG purified from Sj?gren sera mediate apoptosis of the A-253 cell line in a caspase-dependent manner.     

Uropathogenic Escherichia coli induces extrinsic and intrinsic cascades to initiate urothelial apoptosis

A murine model of urinary tract infection identified urothelial apoptosis as a key event in the pathogenesis mediated by uropathogenic Escherichia coli (UPEC), yet the mechanism of this important host response is not well characterized. We employed a culture model of UPEC-urothelium interactions to examine the biochemical events associated with urothelial apoptosis induced by the UPEC strain NU14. NU14 induced DNA cleavage within 5 h that was inhibited by the broad caspase inhibitor ZVAD, and urothelial caspase 3 activity was induced within 3 h of exposure to type 1 piliated NU14 and was dependent upon interactions mediated by the type 1 pilus adhesin FimH. Flow cytometry experiments using chloromethyl-X-rosamine and Indo-1 revealed FimH-dependent mitochondrial membrane depolarization and elevated [Ca(2+)](in), respectively, indicating activation of the intrinsic apoptotic pathway. Consistent with this possibility, overexpression of Bcl(XL) inhibited NU14 activation of caspase 3. Immunoblotting, caspase inhibitors, and caspase activity assays implicated both caspase 2 and caspase 8 in apoptosis, suggesting the involvement of the intrinsic and extrinsic apoptotic cascades. To reconcile the apparent activation of both extrinsic and intrinsic pathways, we examined Bid-green fluorescent protein localization and observed translocation from the cytosol to mitochondria in response to either NU14 or purified FimH. These data suggest that FimH acts as a tethered toxin of UPEC that activates caspase-dependent urothelial apoptosis via direct induction of the extrinsic pathway and that the intrinsic pathway is activated indirectly as a result of coupling by caspase 8-mediated Bid cleavage.     

Involvement of NF-kappaB and mitochondrial pathways in docetaxel-induced apoptosis of human oral squamous cell carcinoma

Apoptosis induced by docetaxel that interferes with microtubule polymerization dynamics and is used clinically to treat advanced cancers, has not been fully defined in squamous cell carcinoma. In this study, apoptotic events involved in docetaxel treatment were investigated. When the human oral squamous cell carcinoma cell line HSC-3 was exposed to docetaxel for 72 h, a dose-dependent effect was observed in apoptosis using the TUNEL method. We observed activation of caspase cascade including activities like caspase-3, -8, and -9. And the pan-caspase inhibitor z-VAD-fmk prevented apoptosis induced by docetaxel (0.1 microM), showing participation of caspases in this process. Since an antagonistic CD95-antibody (ZB4) exerted no effect on docetaxel-induced apoptosis, CD95/CD95L interaction was not involved in this pathway. The caspase-8-like activity was inhibited not only by IETD-fmk (caspase-8) but also by DEVD-fmk (caspase-3). The results indicate that the caspase-8-like activation occurred downstream of DEVDase. Docetaxel promoted the formation of reactive oxygen species (ROS) in mitochondria, and preincubation of cells with anti-oxidants such as N-acetyl cysteine and pyrrolidine dithiocarbamate, protected against apoptosis mediated by docetaxel. Furthermore, treatment with docetaxel elicited reduction of mitochondrial membrane potential, and release of cytochrome c to cytosol, after 48 h of treatment. We observed binding activity to NF-kappaB consensus site and interference with the mitochondrial function via NF-kappaB after docetaxel treatment. Preventing pro-apoptotic property of NF-kappaB inhibited docetaxel-induced apoptosis. Thus, these results suggest that, following the activation of NF-kappaB by docetaxel, apoptosis is elicited through a mitochondria-dependent pathway.     

Protection against CD95-induced apoptosis by chlamydial infection at a mitochondrial step

Chlamydiae are obligate intracellular bacteria that infect human epithelial and myeloid cells. Previous work has established that chlamydiae are able to protect a cell against apoptosis induced by certain experimentally applied stimuli. Here we provide an analysis of this protective activity against the signal transduction during CD95-induced apoptosis. In HeLa cells overexpressing CD95, infection with Chlamydia trachomatis inhibited the appearance of apoptotic morphology, effector caspase activity, the activation of caspase-9 and -3, and the release of cytochrome c from mitochondria. However, caspase-8-processing and activity (measured as cleavage of Bid) were unaffected by the chlamydial infection. Similarly, infection with the species C. pneumoniae did not prevent the activation of caspase-8 but inhibited the appearance of effector caspase activity upon signaling through CD95. Furthermore, infection with C. trachomatis was able to inhibit CD95-induced apoptosis in Jurkat lymphoid cells, where a mitochondrial contribution is required, but not in SKW6.4 lymphoid cells, where caspase-8 directly activates caspase-3. Taken together, these data show that chlamydial infection can protect cells against CD95-induced apoptosis but only where a mitochondrial signaling step is necessary for apoptotic signal transduction.     

Granzyme B-induced apoptosis requires both direct caspase activation and relief of caspase inhibition

Cytotoxic lymphocytes employ Granzyme B as a potent initiator of apoptosis to cleave and activate effector caspases. Unexpectedly, cells transfected with Bcl-2 were resistant to granzyme B-induced killing, suggesting that a mitochondrial pathway was critical. Utilizing cells expressing a dominant-negative caspase 9, the current study demonstrated that caspase activation via the apoptosome was not required. Indeed, cleavage of caspase 3 to p20 still occurred in Bcl-2-transfectants but processing to p17 was blocked. This blockade was recapitulated by the Inhibitor-of-Apoptosis-Protein XIAP and relieved by Smac/DIABLO. Thus granzyme B mediates direct cleavage of caspase 3 and also activates mitochondrial disruption, resulting in the release of proapoptotic proteins that suppress caspase inhibition. Engagement of both pathways is critical for granzyme-induced killing.     

Inhibition of the protein kinase C pathway promotes anti-CD95-induced apoptosis in Jurkat T cells

The role of the basal activity of the serine/threonine protein kinase, protein kinase C (PKC) in the regulation of anti-CD95-induced apoptosis in Jurkat T cells was investigated. The PKC-specific inhibitor GF 109203X and the proposed cPKC-specific inhibitor Go 6976, in a concentration-dependent manner, increased the percentage of cells undergoing apoptosis induced by anti-CD95 mAb as demonstrated by propidium iodide (PI) staining, TUNEL assay and DNA fragmentation by gel electrophoresis. Furthermore, Go 6976 and GF 109203X abrogated phorbol myristate acetate-induced inhibition of anti-CD95-induced apoptosis. To examine the molecular mechanism by which PKC modulates anti-CD95-induced apoptosis, the effects of Go 6976 on known effector and regulatory molecules of cell death were studied. Increased recruitment of cells undergoing apoptosis was associated with enhanced anti-CD95-induced proteolytic cleavage of the most receptor-proximal cysteine protease caspase-8, subsequent cleavage and activation of the machinery protease caspase-3, and cleavage of the caspase substrates DNA-dependent protein kinase catalytic subunit, poly-(ADP-ribose) polymerase and lamin B1. CD95 and FADD protein levels in Jurkat T cells were not altered by Go 6976 treatment. In addition, Go 6976 did not alter protein levels and subcellular distribution of the anti-apoptotic molecules Bcl-2 and Bcl-xL. These data suggest indirectly that basal PKC activity acts at an early stage in the anti-CD95-induced caspase pathway to attenuate subsequent activation of downstream effector molecules and associated apoptosis in Jurkat T cells.      

Caspase inhibitors induce a switch from apoptotic to proinflammatory signaling in CD95-stimulated T lymphocytes

CD95 is a major apoptosis receptor that induces caspase activation and programmed cell death in susceptible cells. CD95-induced apoptosis can be blocked by peptidic caspase inhibitors such as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or Ile-Glu-Thr-Asp-fluoromethyl ketone. Here we show that stimulation of CD95 in the presence of these inhibitors induces necrosis and expression of various proinflammatory cytokines in primary T lymphocytes, such as TNF-alpha, IFN-gamma and granulocyte/macrophage colony-stimulating factor. In the absence of caspase inhibition CD95 stimulation did not result in cytokine expression, indicating that this proinflammatory signaling pathway is suppressed by active caspases. Further analysis with A3.01 T cells revealed that the proinflammatory signaling activity of CD95 was mediated by MEK/ERK, p38 and NF-kappaB signaling pathways. These findings point to a pivotal role of caspases not only as mediators of apoptosis but also as enzymes that prevent proinflammatory signaling during CD95-induced apoptosis. Moreover, our findings may be useful for the development of novel pharmacological strategies.     

How T lymphocytes switch between life and death

While insufficient cell death of activated T cells can result in autoimmune disorders, elimination of too many T cells can lead to immunodeficiency. Therefore, T lymphocyte fate is highly regulated and requires that cells can switch from an apoptosis-resistant towards an apoptosis-sensitive state. This switch is tightly controlled by various effector molecules. Basically, two separate pathways control the fate of antigen-activated T cells: activation-induced cell death (AICD) and activated T cell autonomous death (ACAD). Autoreactive T lymphocytes are eliminated by restimulation via their T cell receptor (TCR) and undergo AICD involving death receptors (extrinsic pathway). In contrast, ACAD can lead to T cell deletion without TCR restimulation, and is determined by the ratio between anti- and pro-apoptotic Bcl-2 family members at the mitochondria (intrinsic pathway). While the extrinsic and the intrinsic pathway lead to caspase activation, non-caspase proteases (e.g., cathepsins) can be released by the lysosomes and might contribute to AICD as well as to ACAD. Activated T cells poses cell death escape mechanisms which are needed for survival of (memory) T cells, but are deleterious for autoimmune disorders or progression of T cell lymphomas.     

Overexpression of caspase-9 triggers its activation and apoptosis in vitro

Aim

To investigate the consequences of increased expression of caspase-9: 1) whether the caspase-9 overexpression resulted in cell death through apoptosis, 2) whether apoptosis could be triggered in normal and tumor cells, and 3) what is the role of caspase-9 in the process.

Methods

The caspase-9 fused to green fluorescent protein was expressed in primary cultures of anterior pituitary cells and of HeLa tumor cells. The expressed caspase-9 and the number of apoptotic and necrotic cells were determined using fluorescence microscopy.

Results

Overexpression of caspase-9 resulted in cell death of primary pituitary cells and HeLa cells. More than 94% of the cells died of apoptosis, which was triggered by the activation of caspase-9, since the cell deaths were prevented in the presence of caspase-9 specific inhibitor. HeLa cells were about 50% more resistant to apoptosis than pituitary cells.

Conclusions

Caspase-9 overexpression and its activation leads to apoptosis. It occurs both in normal and tumor cells. Since the majority of cancer therapy treatments initiate apoptosis through the caspase-9 activation, the modulation of caspase-9 expression may be exploited in designing new ways to control apoptosis in neurodegenerative or malignant diseases.There are two types of cell death: necrosis and apoptosis. Apoptosis or programmed cell death is a process which controls the number and the quality of cells (1). Caspases are proteases, which have a central role in triggering and executing apoptosis (2,3). The two major pathways of triggering apoptosis are the intrinsic and extrinsic pathway (4). The main intrinsic pathway is characterized by mitochondrial dysfunction, with the release of cytochrome c, activation of caspase-9, and subsequently of caspase-3. The extrinsic pathway is activated at the cell surface through death receptor mediated activation of caspase-8 or caspase-10, followed by caspase-3 activation. This pathway may be amplified by caspase-9 activation (intrinsic pathway). Therefore, caspase-9 is one of the main initiator caspases (5). Like the other members of the caspase family, it is synthesized as an inactive zymogen and is activated through proteolytic processing. There are two main pathways for caspase-9 activation: within the apoptosome, a large protein complex, which consists of caspase-9, cytochrome c, and Apaf-1 (6-8), or by proteolytic cleavage by a previously activated caspase, which involves its dimerization (9-11).Among other processes, caspase-9 triggers apoptosis early in the development of nervous system (12,13). Failure of caspase-9 activation is also associated with the resistance for apoptosis in testicular tumors (14). In addition, the gene for caspase-9 maps to the locus disrupted in human tumors, a hotspot for loss of heterozygosity in several cancers, including neuroblastomas (15). A number of initial studies supported the claim that cancer therapy triggered apoptosis by activating the extrinsic pathway of apoptosis (16). Subsequently, there is compelling evidence that the majority of cytotoxic drugs initiate cell death by triggering the cytochrome c/Apaf-1/Caspase-9-dependent pathway through the mitochondria (intrinsic pathway) (16). Embryonic stem cells with disrupted caspase-9 and thymocytes with disrupted Apaf-1 are resistant to cytotoxic drugs, but sensitive to death receptor triggering (extrinsic pathway) (13). In contrast, embryonic fibroblasts disrupted with caspase-8 are sensitive to cytotoxic drugs (17). The relative contributions of intrinsic and extrinsic pathways to apoptosis induced by cytotoxic drugs may also depend on the drug, dose, and kinetics (16). While there are data on the consequences of reduced amounts of caspase-9 in the cells, its overexpression has not been systematically investigated so far.Here we report that caspase-9 overexpression triggers the activation of caspase-9 and apoptosis. It is likely that caspase-9 autoactivates itself under these conditions, since the caspase-9 specific inhibitor prevents the cell deaths. The apoptosis due to overexpression seems to be a ubiquitous process, since it occurs in normal and in tumor transformed cells. HeLa cells are only 50% less sensitive to cell death due to caspase-9 overexpression than the primary pituitary cells.     

Functional expression of CD134 by neutrophils

CD134 (OX40) is a member of the tumor necrosis factor (TNF) receptor superfamily expressed on activated T cells. Here, we show that human peripheral blood neutrophils express CD134. Activation of CD134 by soluble CD134 ligand (OX40 ligand/gp34) resulted in delayed caspase-3 activation and consequently in delayed neutrophil apoptosis in vitro. Moreover, CD134 ligand, like G-CSF, maintained anti-apoptotic Mcl-1 levels and inhibited cleavage of the pro-apoptotic Bcl-2 family members Bid and Bax in these cells, suggesting that CD134-mediated signals block apoptosis pathways proximal to mitochondria activation. In conclusion, CD134 regulates neutrophil survival, suggesting that this molecule does not only contribute to adaptive but also to innate immune responses.