Protein kinase C (PKC) inhibits fas receptor-induced apoptosis through modulation of the loss of K+ and cell shrinkage. A role for PKC upstream of caspases

Cell shrinkage and loss of intracellular K(+) are early requisite features for the activation of effector caspases and apoptotic nucleases in Fas receptor-mediated apoptosis of Jurkat cells, although the mechanisms responsible for both process remain unclear (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). We have now investigated the role of protein kinase C (PKC)-dependent signaling in the regulation of Fas-induced cell shrinkage and loss of K(+) during apoptosis. Anti-Fas induced cell shrinkage was blocked during PKC stimulation by the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (PMA) and by bryostatin-1. Conversely, inhibition of PKC with G?6976, enhanced the anti-Fas-mediated loss of cell volume. Analyses of mitochondrial membrane potential and DNA fragmentation revealed that the PKC-mediated effect observed in cell volume is propagated to these late features of apoptosis. Flow cytometric analyses and (86)Rb efflux experiments revealed that a primary effect of PKC appears to be on the modulation of Fas-induced K(+) efflux, since both PMA and bryostatin-1 inhibited extrusion of K(+) that occurs during Fas-mediated cell death, and G?6976 exacerbated the effect of anti-Fas. Interestingly, high extracellular K(+) significantly blocked the effect of anti-Fas alone or anti-Fas combined with G?6976, suggesting an underlying effect of PKC on K(+) loss. Western blot analyses showed the caspase-dependent proteolysis of PKC isotypes delta, epsilon, and theta in whole cell extracts from anti-Fas treated Jurkat T cells. However, stimulation of PKC by PMA or bryostatin-1 prevented this isotypic-specific PKC cleavage during apoptosis, providing further evidence that PKC itself exerts an upstream signal in apoptosis and controls the caspase-dependent proteolytic degradation of PKC isotypes. Finally, we show that PMA or bryostatin-1 prevents the activation of caspase-3 and caspase-8. Thus, this study shows that the protective effect that PKC stimulation exerts in the Fas-mediated apoptotic pathway occurs at a site upstream of caspases-3 and -8.     

Caspase independent/dependent regulation of K(+), cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis.

The loss of cell volume is a fundamental feature of apoptosis. We have previously shown that DNA degradation and caspase activity occur only in cells which have shrunken as a result of potassium and sodium efflux (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). Furthermore, maintaining a normal intracellular potassium concentration represses the cell death process by inhibiting the activity of apoptotic nucleases and suppressing the activation of effector caspases (Hughes, F. M., Jr., Bortner, C. D. Purdy, G. D., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 30567-30576). We have now investigated the relationship between cell shrinkage, ion efflux, and changes in the mitochondrial membrane potential, in addition to the role of caspases in these apoptotic events. Treatment of Jurkat cells with a series of inducers which act via distinct signal transduction pathways, resulted in all of the cell death characteristics including loss of cell viability, cell shrinkage, K(+) efflux, altered mitochondrial membrane potential, and DNA fragmentation. Interestingly, only cells which shrunk had a loss of mitochondrial membrane potential and the other apoptotic characteristics. Treatment of Jurkat cells with an anti-Fas antibody in the presence of the general caspase inhibitor z-VAD, abrogated these features. In contrast, when Jurkat cells were treated with either the calcium ionophore A23187 or thapsigargin, z-VAD failed to prevent cell shrinkage, K(+) efflux, or changes in the mitochondrial membrane potential, while effectively inhibiting DNA degradation. Treatment of Jurkat cells with various apoptotic agents in the presence of either the caspase-3 inhibitor DEVD, or the caspase-8 inhibitor IETD also blocked DNA degradation, but failed to prevent other characteristics of apoptosis. Together these data suggest that the cell shrinkage, K(+) efflux, and changes in the mitochondrial membrane potential are tightly coupled, but occur independent of DNA degradation, and can be largely caspase independent depending on the particular signal transduction pathway.     

Sequential caspase-2 and caspase-8 activation upstream of mitochondria during ceramideand etoposide-induced apoptosis

Recently, caspase-2 was shown to act upstream of mitochondria in stress-induced apoptosis. Activation of caspase-8, a key event in death receptor-mediated apoptosis, also has been demonstrated in death receptor-independent apoptosis. The regulation of these initiator caspases, which trigger the mitochondrial apoptotic pathway, is unclear. Here we report a potential regulatory role of caspase-2 on caspase-8 during ceramide-induced apoptosis. Our results demonstrate the sequential events of initiator caspase-2 and caspase-8 activation, Bid cleavage and translocation, and mitochondrial damage followed by downstream caspase-9 and -3 activation and cell apoptosis after ceramide induction in T cell lines. The expression of truncated Bid (tBid) and the reduction in mitochondrial transmembrane potential were blocked by caspase-2 or caspase-8, but not caspase-3, knockdown using an RNA interference technique. Ceramide-induced caspase-8 activation, mitochondrial damage, and apoptosis were blocked in caspase-2 short interfering RNA-expressing cells. Therefore, caspase-2 acts upstream of caspase-8 during ceramide-induced mitochondrial apoptosis. Similarly, sequential caspase-2 and caspase-8 activation upstream of mitochondria was also observed in etoposide-induced apoptosis. These data suggest sequential initiator caspase-2 and caspase-8 activation in the mitochondrial apoptotic pathway induced by ceramide or etoposide.     

Caspase-8, c-FLIP, and caspase-9 in c-Myc-induced apoptosis of fibroblasts

c-Myc is known to induce or potentiate apoptotic processes predominantly by triggering or enhancing the activity of caspases, but the activation mechanisms of caspases by c-Myc remain still poorly understood. Here we found that in MycER™ rat fibroblasts the activation of c-Myc led to an early activation and cleavage of the initiator caspase-8, and concurrent processing and activation of the effector caspases 3 and 7. Interestingly, the expression of cellular FLICE inhibitory protein (c-FLIP) mRNA and the encoded protein, c-FLIP_L, a catalytically inactive homologue of caspase-8, were down-regulated prior to or coincidently with the activation of caspase-8. Of the other known initiators, caspase-9, involved in the mitochondrial pathway, was activated/processed surprisingly late, only after the effector caspases 3/7. Further, we studied the potential involvement of the Fas- and tumor necrosis factor receptor (TNFR)-mediated signaling in the activation of caspase-8 by c-Myc. Blocking of the function of these death receptors by neutralizing antibodies against Fas ligand and TNF-α did not prevent the processing of caspase-8 or cell death. c-Myc was neither found to induce any changes in the expression of TNF-related apoptosis inducing ligand (TRAIL) or its receptor. These data suggest that caspase-8 does not become activated through an extrinsic but an “intrinsic/intracellular” apoptotic pathway unleashed by the down-regulation of c-FLIP by c-Myc. Moreover, ectopic expression of c-FLIP_L inhibited the c-Myc-induced apoptosis.     

Protein kinase C regulates FADD recruitment and death-inducing signaling complex formation in Fas/CD95-induced apoptosis

Activation of protein kinase C (PKC) triggers cellular signals that inhibit Fas/CD95-induced cell death in Jurkat T-cells by poorly defined mechanisms. Previously, we have shown that one effect of PKC on Fas/CD95-dependent cell death occurs through inhibition of cell shrinkage and K(+) efflux (Gómez-Angelats, M., Bortner, C. D., and Cidlowski, J. A. (2000) J. Biol. Chem. 275, 19609-19619). Here we report that PKC alters Fas/CD95 signaling from the plasma membrane to the activation of caspases by exerting a profound action on survival/cell death decisions. Specific activation of PKC with 12-O-tetradecanoylphorbol-13-acetate or bryostatin-1 induced translocation of PKC from the cytosol to the membrane and effectively inhibited cell shrinkage and cell death triggered by anti-Fas antibody in Jurkat cells. In contrast, inhibition of classical PKC isotypes with G?6976 exacerbated the effect of Fas activation on both apoptotic volume decrease and cell death. PKC activation/inhibition did not affect anti-Fas antibody binding to the cell surface, intracellular levels of FADD (Fas-associated protein with death domain), or c-FLIP (cellular FLICE-like inhibitory protein) expression. However, processing/activation of both caspase-8 and caspase-3 and BID cleavage were markedly blocked upon PKC activation and, conversely, were augmented during PKC inhibition, suggesting a role for PKC upstream of caspase-8 processing and activation. Analysis of death-inducing signaling complex (DISC) formation was carried out to examine the influence of PKC on recruitment of both FADD and procaspase-8 to the Fas receptor. PKC activation blocked FADD recruitment and caspase-8 activation and thus DISC formation in both type I and II cells. In contrast, inhibition of classical PKCs promoted the opposite effect on the Fas pathway by rapidly increasing FADD recruitment, caspase-8 activation, and DISC formation. Together, these data show that PKC finely modulates Fas/CD95 signaling by altering the efficiency of DISC formation.     

Delineation of the caspase-9 signaling cascade

In the intrinsic apoptosis pathway, mitochondrial disruption leads to the release of multiple apoptosis signaling molecules, triggering both caspase-dependent and -independent cell death. The release of cytochrome c induces the formation of the apoptosome, resulting in caspase-9 activation. Multiple caspases are activated downstream of caspase-9, however, the precise order of caspase activation downstream of caspase-9 in intact cells has not been completely resolved. To characterize the caspase-9 signaling cascade in intact cells, we employed chemically induced dimerization to activate caspase-9 specifically. Dimerization of caspase-9 led to rapid activation of effector caspases, including caspases-3, -6 and -7, as well as initiator caspases, including caspases-2, -8 and -10, in H9 and Jurkat cells. Knockdown of caspase-3 suppressed caspase-9-induced processing of the other caspases downstream of caspase-9. Silencing of caspase-6 partially inhibited caspase-9-mediated processing of caspases-2, -3 and -10, while silencing of caspase-7 partially inhibited caspase-9-induced processing of caspase-2, -3, -6 and -10. In contrast, deficiency in caspase-2, -8 or -10 did not significantly affect the caspase-9-induced caspase cascade. Our data provide novel insights into the ordering of a caspase signaling network downstream of caspase-9 in intact cells during apoptosis.     

Caspase-8 and Apaf-1-independent caspase-9 activation in Sendai virus-infected cells

Apoptotic cell death is of central importance in the pathogenesis of viral infections. Activation of a cascade of cysteine proteases, i.e. caspases, plays a key role in the effector phase of virus-induced apoptosis. However, little is known about pathways leading to the activation of initiator caspases in virus-infected host cells. Recently, we have shown that Sendai virus (SeV) infection triggers apoptotic cell death by activation of the effector caspase-3 and initiator caspase-8. We now investigated mechanisms leading to the activation of another initiator caspase, caspase-9. Unexpectedly we found that caspase-9 cleavage is not dependent on the presence of active caspases-3 or -8. Furthermore, the presence of caspase-9 in mouse embryonic fibroblast (MEF) cells was a prerequisite for Sendai virus-induced apoptotic cell death. Caspase-9 activation occurred without the release of cytochrome c from mitochondria and was not dependent on the presence of Apaf-1 or reactive oxygen intermediates. Our results therefore suggest an alternative mechanism for caspase-9 activation in virally infected cells beside the well characterized pathways via death receptors or mitochondrial cytochrome c release.     

Renal cell carcinoma tumors induce T cell apoptosis through receptor-dependent and receptor-independent pathways

Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.     

Caspase activation throughout the first wave of spermatogenesis in the rat

Early in postnatal life, the first wave of spermatogenesis is accompanied by an initial wave of germ cell apoptosis. This may reflect an adjustment in the number of germ cells that can be adequately maintained by Sertoli cells. Two major pathways (intrinsic and extrinsic) are involved in the process of caspase activation and apoptosis in mammalian cells. The extrinsic pathway is characterized by the oligomerization of death receptors such as FAS or tumor necrosis factor, followed by the activation of caspase-8 and caspase-3. The intrinsic pathway involves the activation of procaspase-9, which in turn activates caspase-3. Extensive information is available concerning apoptotic inducers and their possible mechanisms in the adult rat. However, no data exist regarding the molecular and cellular mechanisms governing physiological cell death during puberty in the male rat. We have studied caspase activation throughout the first wave of spermatogenesis in the rat under physiological conditions, by combining the TUNEL procedure with the localization of active caspases in germ cells. We observed TUNEL-positive germ cells in rats of 5–40 days of age, the highest number being found in 25-day-old rats. TUNEL-positive and caspase-3-positive germ cells appeared as long chains of interconnected germ cells in 25-day-old rats. Caspase activation was assayed by either immunohistochemistry with antibodies against active caspase-3, -8, and -9, or by determining enzymatic activity in seminiferous tubules extracts. Both techniques showed activation of caspase-3, -8, and -9 in 25-day-old rats and low enzymatic activity at other ages. Confocal scanning laser microscopy indicated that active caspase-3, -8, and -9 co-localized with TUNEL-positive cells. Thus, caspase-3, -8, and -9 are active in apoptotic germ cells during the first wave of rat spermatogenesis. The extrinsic pathway of apoptosis may therefore play an important role in germ cell apoptosis during puberty in the rat.This work was financed by a research grant from FONDECYT (1040800) to R.D.M.     

Activation of intrinsic and extrinsic pathways in apoptotic signaling during UV-C-induced death of Jurkat cells: the role of caspase inhibition

We have examined UV irradiation-induced cell death in Jurkat cells and evaluated the relationships that exist between inhibition of caspase activity and the signaling mechanisms and pathways of apoptosis. Jurkat cells were irradiated with UV-C light, either with or without pretreatment with the pan-caspase inhibitor, z-VAD-fmk (ZVAD), or the more selective caspase inhibitors z-IETD-fmk (IETD), z-LEHD-fmk (LEHD), and z-DEVD-fmk (DEVD). Flow cytometry was used to examine alterations in viability, cell size, plasma membrane potential (PMP), mitochondrial membrane potential (DeltaPsi(mito)), intracellular Na(+) and K(+) concentrations, and DNA degradation. Processing of pro-caspases 3, 8, and 9 and the pro-apoptotic protein Bid was determined by Western blotting. UV-C irradiation of Jurkat cells resulted in characteristic apoptosis within 6 h after treatment and pretreatment of cells with ZVAD blocked these features. In contrast, pretreatment of the cells with the more selective caspase inhibitors under conditions that effectively blocked DNA degradation and inhibited caspase 3 and 8 processing as well as Bid cleavage had little protective effect on the other apoptotic characteristics examined. Thus, both intrinsic and extrinsic pathways are activated during UV-induced apoptosis in Jurkat cells and this redundancy appears to assure cell death during selective caspase inhibition.     

Regulation of constitutive neutrophil apoptosis by the alpha,beta-unsaturated aldehydes acrolein and 4-hydroxynonenal

Reactive alpha,beta-unsaturated aldehydes are major components of common environmental pollutants and are products of lipid oxidation. Although these aldehydes have been demonstrated to induce apoptotic cell death in various cell types, we recently observed that the alpha,beta-unsaturated aldehyde acrolein (ACR) can inhibit constitutive apoptosis of polymorphonuclear neutrophils and thus potentially contribute to chronic inflammation. The present study was designed to investigate the biochemical mechanisms by which two representative alpha,beta-unsaturated aldehydes, ACR and 4-hydroxynonenal (HNE), regulate neutrophil apoptosis. Whereas low concentrations of either aldehyde (<10 microM) mildly promoted apoptosis in neutrophils (reflected by increased phosphatidylserine exposure, caspase-3 activation, and mitochondrial cytochrome c release), higher concentrations prevented critical features of apoptosis (caspase-3 activation, phosphatidylserine exposure) and caused delayed neutrophil cell death with characteristics of necrosis/oncosis. Inhibition of caspase-3 activation by either aldehyde occurred despite increases in mitochondrial cytochrome c release and occurred in close association with depletion of cellular GSH and with cysteine modifications within caspase-3. However, procaspase-3 processing was also prevented, because of inhibited activation of caspases-9 and -8 under similar conditions, suggesting that ACR (and to a lesser extent HNE) can inhibit both intrinsic (mitochondria dependent) and extrinsic mechanisms of neutrophil apoptosis at initial stages. Collectively, our results indicate that alpha,beta-unsaturated aldehydes can inhibit constitutive neutrophil apoptosis by common mechanisms, involving changes in cellular GSH status resulting in reduced activation of initiator caspases as well as inactivation of caspase-3 by modification of its critical cysteine residue.     

Different roles of spermine in glucocorticoid- and Fas-induced apoptosis

Two experimental systems representative of the mitochondrial and death receptor apoptotic pathways are the dexamethasone-induced programmed cell death in mouse thymocytes and the antibody-mediated cross-ligation of the Fas receptor in the human leukemic T-cell line Jurkat, respectively. In both cell systems, caspase-9, -8, and -3 were activated upon induction of apoptosis and a sub-G(1) peak appeared as a sign of ongoing DNA fragmentation. Addition of 1 mM spermine together with dexamethasone inhibited caspase activation and the appearance of the sub-G(1) peak in mouse thymocytes. In contrast, Fas-induced cell death was totally unaffected by spermine addition. Spermine addition significantly elevated the spermine concentration in both thymocytes and Jurkat cells. Thus, spermine per se did not inhibit the caspases but rather their activation. The fact that spermine inhibited caspase activation only in the thymocytes implies that spermine inhibited dexamethasone-induced apoptosis upstream of caspase-9 activation.     

p53-dependent Fas expression is critical for Ginsenoside Rh2 triggered caspase-8 activation in HeLa cells

We have recently reported that Ginsenoside Rh2 (G-Rh2) induces the activation of two initiator caspases, caspase-8 and caspase-9 in human cancer cells. However, the molecular mechanism of its death-inducing function remains unclear. Here we show that G-Rh2 stimulated the activation of both caspase-8 and caspase-9 simultaneously in HeLa cells. Under G-Rh2 treatment, membrane death receptors Fas and TNFR1 are remarkably upregulated. However, the induced expression of Fas but not TNFR1 was contributed to the apoptosis process. Moreover, significant increases in Fas expression and caspase-8 activity temporally coincided with an increase in p53 expression in p53-nonmutated HeLa and SK-HEP-1 cells upon G-Rh2 treatment. In contrast, Fas expression and caspase-8 activity remained constant with G-Rh2 treatment in p53-mutated SW480 and PC-3 cells. In addition, siRNA-mediated knockdown of p53 diminished G-Rh2-induced Fas expression and caspase-8 activation. These results indicated that G-Rh2-triggered extrinsic apoptosis relies on p53-mediated Fas over-expression. In the intrinsic apoptotic pathway, G-Rh2 induced strong and immediate translocation of cytosolic BAK and BAX to the mitochondria, mitochondrial cytochrome c release, and subsequent caspase-9 activation both in HeLa and in SW480 cells. p53-mediated Fas expression and subsequent downstream caspase-8 activation as well as p53-independent caspase-9 activation all contribute to the activation of the downstream effector caspase-3/-7, leading to tumor cell death. Taken together, we suggest that G-Rh2 induces cancer cell apoptosis in a multi-path manner and is therefore a promising candidate for antitumor drug development.     

Antimycin A-induced killing of HL-60 cells: apoptosis initiated from within mitochondria does not necessarily proceed via caspase 9.

BACKGROUND: Antimycin A (AMA) inhibits mitochondrial electron transport, collapses the mitochondrial membrane potential, and causes the production of reactive oxygen species. Previous work by me and my colleagues has demonstrated that AMA causes an array of typical apoptotic phenomena in HL-60 cells. The hypothesis that AMA causes HL-60 apoptosis by the intrinsic apoptotic pathway has now been tested. METHODS: Z-LEHD-FMK and Z-IETD-FMK were used as specific inhibitors of the initiator caspases 9 and 8, respectively. Caspase 3 activation, DNA fragmentation, and cellular disintegration were measured by flow cytometry. Cytochrome c release, chromatin condensation, and nuclear fragmentation were measured by microscopy. RESULTS: AMA caused mitochondrial cytochrome c release and neither Z-LEHD-FMK nor Z-IETD-FMK inhibited that. In the absence of caspase inhibition there was a very close correlation between cytochrome c release and caspase 3 activation. Z-LEHD-FMK blocked caspase 3 activation but enhanced DNA fragmentation and failed to stop nuclear or cellular disintegration. Z-IETD-FMK also blocked caspase 3 activation but, in contrast to Z-LEHD-FMK, delayed DNA fragmentation and disintegration of the nucleus and the cell. CONCLUSIONS: The hypothesis to explain AMA-induced HL-60 apoptosis was clearly inadequate because: (a) caspase 9 inhibition did not prevent DNA fragmentation or cell death, (b) apoptosis proceeded in the absence of caspase-3 activation, (c) the main pathway leading to activation of the executioner caspases was by caspase-8 activation, but caspase 8 inhibition only delayed apoptosis, and (d) activation of caspases 8 and 9 may be necessary for caspase-3 activation. Thus, in this cell model, apoptosis triggered from within the mitochondria does not necessarily proceed by caspase 9, and caspase 3 is not critical to apoptosis. The results provide further evidence that, when parts of the apoptotic network are blocked, a cell is able to complete the program of cell death by alternate pathways.     

X Chromosome-linked Inhibitor of Apoptosis Regulates Cell Death Induction by Proapoptotic Receptor Agonists

Proapoptotic receptor agonists cause cellular demise through the activation of the extrinsic and intrinsic apoptotic pathways. Inhibitor of apoptosis (IAP) proteins block apoptosis induced by diverse stimuli. Here, we demonstrate that IAP antagonists in combination with Fas ligand (FasL) or the death receptor 5 (DR5) agonist antibody synergistically stimulate death in cancer cells and inhibit tumor growth. Single-agent activity of IAP antagonists relies on tumor necrosis factor-α signaling. By contrast, blockade of tumor necrosis factor-α does not affect the synergistic activity of IAP antagonists with FasL or DR5 agonist antibody. In most cancer cells, proapoptotic receptor agonist-induced cell death depends on amplifying the apoptotic signal via caspase-8-mediated activation of Bid and subsequent activation of the caspase-9-dependent mitochondrial apoptotic pathway. In the investigated cancer cell lines, induction of apoptosis by FasL or DR5 agonist antibody can be inhibited by knockdown of Bid. However, knockdown of X chromosome-linked IAP (XIAP) or antagonism of XIAP allows FasL or DR5 agonist antibody to induce activation of effector caspases efficiently without the need for mitochondrial amplification of the apoptotic signal and thus rescues the effect of Bid knockdown in these cells.     

Identification of early intermediates of caspase activation using selective inhibitors and activity-based probes

Caspases are cysteine proteases that are key effectors in apoptotic cell death. Currently, there is a lack of tools that can be used to monitor the regulation of specific caspases in the context of distinct apoptotic programs. We describe the development of highly selective inhibitors and active site probes and their applications to directly monitor executioner (caspase-3 and -7) and initiator (caspase-8 and -9) caspase activity. Specifically, these reagents were used to dissect the kinetics of caspase activation upon stimulation of apoptosis in cell-free extracts and intact cells. These studies identified a full-length caspase-7 intermediate that becomes catalytically activated early in the pathway and whose further processing is mediated by mature executioner caspases rather than initiator caspases. This form also shows distinct inhibitor sensitivity compared to processed caspase-7. Our data suggest that caspase-7 activation proceeds through a previously uncharacterized intermediate that is formed without cleavage of the intact zymogen.     

Radiation induces apoptosis primarily through the intrinsic pathway in mammalian cells

Radiation-induced tumor cells death is the theoretical basis of tumor radiotherapy. Death signaling disorder is the most important factor for radioresistance. However, the signaling pathway(s) leading to radiation-triggered cell death is (are) still not completely known. To better understand the cell death signaling induced by radiation, the immortalized mouse embryonic fibroblast (MEF) deficient in “initiator” caspases, “effector” caspases or different Bcl-2 family proteins together with human colon carcinoma cell HCT116 were used. Our data indicated that radiation selectively induced the activation of caspase-9 and caspase-3/7 but not caspase-8 by triggering mitochondrial outer membrane permeabilization (MOMP). Importantly, the role of radiation in MOMP is independent of the activation of both “initiator” and “effector” caspases. Furthermore, both proapoptotic and antiapoptotic Bcl-2 family proteins were involved in radiation-induced apoptotic signaling. Overall, our study indicated that radiation specifically triggered the intrinsic apoptotic signaling pathway through Bcl-2 family protein-dependent mitochondrial permeabilization, which indicates targeting mitochondria is a promising strategy for cancer radiotherapy.     

Caspase-10-dependent cell death in Fas/CD95 signalling is not abrogated by caspase inhibitor zVAD-fmk

Background

Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated.

Methodology and Principal Findings

The present study shows that FasL-induced cell death was completely impaired in caspase-8- and caspase-10-doubly deficient (I9-2e) Jurkat leukaemia T-cell lines. Over-expressing of either caspase-8 or caspase-10 in I9-2e cells triggered cell death and restored sensitivity to FasL, further arguing for a role of both initiator caspases in Fas apoptotic signalling. In the presence of zVAD-fmk, FasL triggered an alternative form of cell death similarly in wild-type (A3) and in caspase-8-deficient Jurkat cells expressing endogenous caspase-10 (clone I9-2d). Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death. Noteworthy, ectopic expression of caspase-10 in I9-2e and HeLa cells restored the ability of FasL to trigger cell death in the presence of zVAD-fmk. As a matter of fact, FasL-triggered caspase-10 processing still occurred in the presence of zVAD-fmk.

Conclusions and Significance

Altogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling. Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.     

Immunofluorescent detection of activation of initiator caspases-8 and -9 during pharmacologically induced apoptosis of cultured HeLa and endothelial cells

Caspases, a group of cysteine-activated aspartate-directed proteases, play an integral role in the execution of programmed cell death or apoptosis. In the cellular caspase cascade, the processing of native proenzymes into activated forms of downstream, effector caspases is dependent on the activation of initiator caspases-8 and -9. We describe a staining procedure for immunofluorescence-based analysis of activation of caspase-8 and -9 during pharmacologically induced apoptosis in primary cultures of human umbilical vein-derived endothelial cells and in an established line of HeLa cells. Using cleavage site-directed antibodies, specific intracellular detection for cleaved fragments of caspase-8 and -9 was accomplished during apoptosis induced by staurosporine and etoposide. The population of cells displaying morphological signs of apoptosis, evidence for DNA strand breaks by TUNEL analysis, and positive staining for active forms of caspase-8 and caspase-9 increased with the duration of treatment, suggesting activation of initiator caspases in correlation with the onset and progression of apoptosis. The application of immunocytochemical staining procedures for quick and specific in situ detection may effectively aid the identification of participating upstream caspases and elucidation of complex apoptosis signaling mechanisms.     

FLIP inhibits endothelial cell apoptosis during hyperoxia by suppressing Bax

High oxygen tension (hyperoxia) causes pulmonary cell death, involving apoptosis, necrosis, or mixed death phenotypes, though the underlying mechanisms remain unclear. In mouse lung endothelial cells (MLEC) hyperoxia activates both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) apoptotic pathways. We examined the hypothesis that FLIP, an inhibitor of caspase-8, can protect endothelial cells against the lethal effects of hyperoxia. Hyperoxia caused the time-dependent downregulation of FLIP in MLEC. Overexpression of FLIP attenuated intracellular reactive oxygen species generation during hyperoxia exposure, by downregulating extracellular-regulated kinase-1/2 activation and p47(phox) expression. FLIP prevented hyperoxia-induced trafficking of the death-inducing signal complex from the Golgi apparatus to the plasma membrane. Furthermore, FLIP blocked the activations of caspase-8/Bid, caspases -3/-9, and inhibited the mitochondrial translocation and activation of Bax, resulting in protection against hyperoxia-induced cell death. Under normoxic conditions, FLIP expression increased the phosphorylation of p38 mitogen-activated protein kinase leading to increased phosphorylation of Bax during hyperoxic stress. Furthermore, FLIP expression markedly inhibited protein kinase C activation and expression of distinct protein kinase C isoforms (alpha, eta, and zeta), and stabilized an interaction of PKC with Bax. In conclusion, FLIP exerted novel inhibitory effects on extrinsic and intrinsic apoptotic pathways, which significantly protected endothelial cells from the lethal effects of hyperoxia.