Spontaneous human monocyte apoptosis utilizes a caspase-3-dependent pathway that is blocked by endotoxin and is independent of caspase-1.

Apoptosis is an important mechanism for regulating the numbers of monocytes and macrophages. Caspases (cysteine-aspartate-specific proteases) are key molecules in apoptosis and require proteolytic removal of prodomains for activity. Caspase-1 and caspase-3 have both been connected to apoptosis in other model systems. The present study attempted to delineate what role these caspases play in spontaneous monocyte apoptosis. In serum-free conditions, monocytes showed a commitment to apoptosis as early as 4 h in culture, as evidenced by caspase-3-like activity. Apoptosis, as defined by oligonucleosomal DNA fragmentation, was prevented by a generalized caspase inhibitor, z-VAD-FMK, and the more specific caspase inhibitor, z-DEVD-FMK. The caspase activity was specifically attributable to caspase-3 by the identification of cleavage of procaspase-3 to active forms by immunoblots and by cleavage of the fluorogenic substrate DEVD-AFC. In contrast, a caspase-1 family inhibitor, YVAD-CMK, did not protect monocytes from apoptosis, and the fluorogenic substrate YVAD-AFC failed to show an increase in activity in apoptotic monocytes. When cultured with LPS (1 microgram/ml), monocyte apoptosis was prevented, as was the activation of caspase-3. Unexpectedly, LPS did not change baseline caspase-1 activity. These findings link spontaneous monocyte apoptosis to the proteolytic activation of caspase-3.     

Protein kinase C inhibition induces DNA fragmentation in COLO 205 cells which is blocked by cysteine protease inhibition but not mediated through caspase-3

Enhancing apoptosis to remove abnormal cells has potential in reversing cancerous processes. Caspase-3 activation generally accompanies apoptosis and its substrates include enzymes responsible for DNA fragmentation and isozymes of protein kinase C (PKC). Recent data, however, question its obligatory role in apoptosis. We have examined whether modulation of PKC activity induces apoptosis in COLO 205 cells and the role of caspase-3. Proliferation ([3H]thymidine) and apoptosis (DNA fragmentation and FACS) of COLO 205 cells were measured in response to PKC activation and inhibition. Caspase-3 activity was assayed and the effects of its inhibition with Ac-DEVD-cmk, and the effect of other protease inhibitors, on apoptosis were determined. PKC activation and inhibition both reduced DNA synthesis and induced DNA fragmentation. As PKC inhibitors induced DNA fragmentation more rapidly than PKC activators and failed to block activator effects, we conclude that it is PKC down-regulation (i.e., inhibition) after activator exposure that mediates apoptosis. Increases in caspase-3 activity occurred during apoptosis but apoptosis was not blocked by caspase inhibition. By contrast, the cysteine protease inhibitor, E-64d, blocked apoptosis. Cysteine proteases not of the caspase family may either act more closely to the apoptotic process than caspases or lie on an alternative, more active pathway.     

The proteolytic procaspase activation network: an in vitro analysis

In general, apoptotic stimuli lead to activation of caspases. Once activated, a caspase can induce intracellular signaling pathways involving proteolytic activation of other caspase family members. We report the in vitro processing of eight murine procaspases by their enzymatically active counterparts. Caspase-8 processed all procaspases examined. Caspase-1 and -11 processed the effector caspases procaspase-3 and -7, and to a lesser extent procaspase-6. However, vice versa, none of the caspase-1-like procaspases was activated by the effector caspases. This suggests that the caspase-1 subfamily members either act upstream of the apoptosis effector caspases or else are part of a totally separate activation pathway. Procaspase-2 was maturated by caspase-8 and -3, and to a lesser extent by caspase-7, while the active caspase-2 did not process any of the procaspases examined, except its own precursor. Hence, caspase-2 might not be able to initiate a wide proteolytic signaling cascade. Additionally, cleavage data reveal not only proteolytic amplification between caspase-3 and -8, caspase-6 and -3, and caspase-6 and -7, but also positive feedback loops involving multiple activated caspases. Our results suggest the existence of a hierarchic proteolytic procaspase activation network, which would lead to a dramatic increase in multiple caspase activities once key caspases are activated. The proteolytic procaspase activation network might allow that different apoptotic stimuli result in specific cleavage of substrates responsible for typical processes at the cell membrane, the cytosol, the organelles, and the nucleus, which characterize a cell dying by apoptosis.     

Caspase-7 gene disruption reveals an involvement of the enzyme during the early stages of apoptosis

Caspases play a key role during apoptotic execution. In an attempt to elucidate the specific role of caspase-7 we generated a chicken DT40 cell line in which both alleles of the gene were disrupted. Viability assays showed that caspase-7-/- clones are more resistant to the common apoptosis-inducing drugs etoposide and staurosporine. Caspase-7-/- cells show a delay in phosphatidylserine externalization and DNA fragmentation as well as cleavage of the caspase substrates poly(ADP-ribose) polymerase 1 and lamins B1 and B2. Caspase affinity labeling and activity assays indicated that deficient cells exhibit a delay in caspase activation compared with wild type DT40 cells, providing an explanation for the differences in apoptotic execution between caspase-7 null and wild type DT40 cells. These results strongly suggest that caspase-7 is involved earlier than other effector caspases in the apoptotic execution process in DT40 B lymphocytes.     

Development of cell death-based method for the selectivity screening of caspase-1 inhibitors

Caspase-1 selective inhibitors are novel therapeutic agents for inflammatory diseases. Selectivity assays for caspases can be initiated with purified enzyme, making these assays very costly and time consuming. Therefore, there is a need to develop a fast and reliable cell-based assay, which can be used for the selectivity screening of multiple caspases in a biologically relevant context in a single assay. In this study, we have developed an assay in which DNA fragmentation, a hallmark of apoptosis, of Jurkat cell line was examined post induction with etoposide in the presence or absence of inhibitors of caspases 1, 3, 8, 9 and pan-caspase inhibitors. We observed that caspases-3, -8, -9 and pan caspase inhibitors resulted in significant inhibition of etoposide-induced DNA fragmentation. However, caspase-1 specific inhibitor failed to prevent DNA fragmentation, suggesting that either caspases belonging to caspase-1 family (1, 4 and 5) are not present in the Jurkat cells or might not be involved in the etoposide-induced DNA fragmentation. Since the inhibition of caspases 3, 8 and 9 is accompanied by the down regulation of the activity of a cascade of caspases (caspases 2, 6, 7, 9 and 10), selectivity of caspase-I inhibitors can be ascertained for the above panel (caspases 2, 6, 7, 8, 9 and 10) of caspases from this single assay.     

Cell death pathways in juvenile Batten disease

Apoptosis, Golgi fragmentation and elevated ceramide levels occur in Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) neurons, lymphoblasts and fibroblasts. Our purpose was to examine whether apoptosis is the mechanism of cell death in JNCL. This was tested by analyzing caspase-dependent/independent pathways and autophagy, and caspase effects on ceramide and Golgi fragmentation. zVAD prevented caspase activation, but not all cell death. Inhibiting caspase-8 suppressed caspases more than inhibition of any other caspase. Inhibiting caspase-8/6 was synergistic. zVAD suppressed autophagy. 3-methyladenine suppressed caspase activation less than zVAD did. Blocking autophagy/caspase-8/or-6 was synergistic. Blocking autophagy/caspase-3/or-9 was not. Inhibiting caspase-9/3 suppressed autophagy. Golgi fragmentation was suppressed by zVAD, and blocked by CLN3. CLN3, not zVAD, prevented ceramide elevation. In conclusion: caspase-dependent/independent apoptosis and autophagy occur caspase-dependent pathways initiate autophagy Golgi fragmentation results from apoptosis ceramide elevation is independent of caspases, and CLN3 blocks all cell death, prevents Golgi fragmentation and elevation of ceramide in JNCL.     

Hydrogen peroxide-induced apoptosis in HL-60 cells requires caspase-3 activation

Apoptosis has been associated with oxidative stress in biological systems. Caspases have been considered to play a pivotal role in the execution phase of apoptosis. However, which caspases function as executioners in reactive oxygen species (ROS)-induced apoptosis is not known. The present study was performed to identify the major caspases acting in ROS-induced apoptosis. Treatment of HL-60 cells with 50 μM hydrogen peroxide (H₂O₂) for 4 h induced the morphological changes such as condensed and/or fragmented nuclei, increase in caspase-3 subfamily protease activities, reduction of the procaspase-3 and a DNA fragmentation. To determine the role of caspases in H₂O₂-induced apoptosis, caspase inhibitors, acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone(Ac-YVAD-cmk), acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and acetyl-Val-Glu-lle-Aspaldehyde (Ac-VEID-CHO), selective for caspase-1 subfamily, caspase-3 subfamily and caspase-6, respectively, were loaded into the cells using an osmotic lysis of pinosomes method. Of these caspase inhibitors, only Ac-DEVD-CHO completely blocked morphological changes, caspase-3 subfamily protease activation and DNA ladder formation in H₂O₂-treated HL-60 cells. This inhibitory effect was dose-dependent. These results suggest that caspase-3, but not caspase-1 is required for commitment to ROS-triggered apoptosis.     

Caspase-10 triggers Bid cleavage and caspase cascade activation in FasL-induced apoptosis

In contrast to caspase-8, controversy exists as to the ability of caspase-10 to mediate apoptosis in response to FasL. Herein, we have shown activation of caspase-10, -3, and -7 as well as B cell lymphoma-2-interacting domain (Bid) cleavage and cytochrome c release in caspase-8-deficient Jurkat (I9-2) cells treated with FasL. Apoptosis was clearly induced as illustrated by nuclear and DNA fragmentation. These events were inhibited by benzyloxycarbonyl-VAD-fluoromethyl ketone, a broad spectrum caspase inhibitor, indicating that caspases were functionally and actively involved. Benzyloxycarbonyl-AEVD-fluoromethyl ketone, a caspase-10 inhibitor, had a comparable effect. FasL-induced cell death was not completely abolished by caspase inhibitors in agreement with the existence of a cytotoxic caspase-independent pathway. In subpopulations of I9-2 cells displaying distinct caspase-10 expression levels, cell sensitivity to FasL correlated with caspase-10 expression. A robust caspase activation, Bid cleavage, and DNA fragmentation were observed in cells with high caspase-10 levels but not in those with low levels. In vitro, caspase-10, as well as caspase-8, could cleave Bid to generate active truncated Bid (p15). Altogether, our data strongly suggest that caspase-10 can serve as an initiator caspase in Fas signaling leading to Bid processing, caspase cascade activation, and apoptosis.     

Possible involvement of cytochrome c release and sequential activation of caspases in ceramide-induced apoptosis in SK-N-MC cells

Ceramide is characterized as a second messenger of apoptosis induced by various agents such as tumor necrosis factor (TNF-alpha), Fas ligand, hydrogen peroxide, heat shock and ionizing radiation. In this study, we investigated the mechanism of ceramide-induced apoptosis using a human neuroblastoma cell line, SK-N-MC. N-Acetyl-sphingosine (C2-ceramide), a cell-permeable ceramide analogue, was able to induce apoptosis in SK-N-MC cells as estimated by DNA fragmentation and chromatin condensation. C2-ceramide-induced DNA fragmentation was blocked by caspase inhibitor (Z-Asp-CH(2)-DCB). An increase in caspase-3 (CPP32)-like protease activity was evident during C2-ceramide-induced apoptosis, suggesting that caspases are involved in this apoptosis. Moreover, enzymatic cleavage of VDVAD-AFC and LEHD-AFC (specific substrates for caspase-2 and -9, respectively) was increased by treatment with C2-ceramide. To elucidate which types of caspase are activated in C2-ceramide-treated cells, we performed Western blot analysis using antibodies against each isoform. Both proforms of caspase-2 and -3 were decreased in response to C2-ceramide in a time-dependent manner. Mitochondrial cytochrome c is also time-dependently released into the cytosol in response to treatment with C2-ceramide. Addition of cytochrome c into the S-100 fractions prepared from SK-N-MC cells could activate caspase-2 in cell-free systems. These results suggest the possibility that cytochrome c released to the cytosol can activate caspases (caspase-9, -3, and -2) during C2-ceramide-induced apoptosis of SK-N-MC cells.     

Caspase-3 mediated feedback activation of apical caspases in doxorubicin and TNF-α induced apoptosis

Aberrant apoptosis has been associated with the development and therapeutic resistance of cancer. Recent studies suggest that caspase deficiency/downregulation is frequently detected in different cancers. We have previously shown that caspase-3 reconstitution significantly sensitized MCF-7 cells to doxorubicin and etoposide. In contrast to the well established role of caspase-3 as an effector caspase, the focus of this study is to delineate caspase-3 induced feedback activation of the apical caspases-2, -8, -9 and -10A in doxorubicin and TNF-α induced apoptosis. Using cell-free systems we show that caspases-9 and 2 are the most sensitive, caspase-8 is less sensitive and caspase-10A is the least sensitive to caspase-3 mediated-cleavage. When apoptosis is induced by doxorubicin or TNF-α in an intact cell model, cleavage of caspases-8 and -9, but not caspase-2, was markedly enhanced by caspase-3. Caspase-3 mediated-feedback and activation of caspase-8 and -9 in MCF-7/C3 cells is further supported by an increase in the cleavage of caspase-8 and 9 substrates and cytochrome c release. These data indicate that, in addition to its function as an effector caspase, caspase-3 plays an important role in maximizing the activation of apical caspases and crosstalk between the two major apoptotic pathways. The significant impact of caspase-3 on both effector and apical caspases suggests that modulation of caspase-3 activity would be a useful approach to overcome drug resistance in clinical oncology. XiaoHe Yang: This work was supported in part by the Career Development Award DAMD17-99-1-9180 from Department of Defense to X.H.Y.     

A novel caspase-2 complex containing TRAF2 and RIP1

The enzymatic activity of caspases is implicated in the execution of apoptosis and inflammation. Here we demonstrate a novel nonenzymatic function for caspase-2 other than its reported proteolytic role in apoptosis. Caspase-2, unlike caspase-3, -6, -7, -9, -11, -12, and -14, is a potent inducer of NF-kappaB and p38 MAPK activation in a TRAF2-mediated way. Caspase-2 interacts with TRAF1, TRAF2, and RIP1. Furthermore, we demonstrate that endogenous caspase-2 is recruited into a large and inducible protein complex, together with TRAF2 and RIP1. Structure-function analysis shows that NF-kappaB activation occurs independent of enzymatic activity of the protease and that the caspase recruitment domain of caspase-2 is sufficient for the activation of NF-kappaB and p38 MAPK. These results demonstrate the inducible assembly of a novel protein complex consisting of caspase-2, TRAF2, and RIP1 that activates NF-kappaB and p38 MAPK through the caspase recruitment domain of caspase-2 independently of its proteolytic activity.     

Caspases induce cytochrome c release from mitochondria by activating cytosolic factors.

We investigated the ability of caspases (cysteine proteases with aspartic acid specificity) to induce cytochrome c release from mitochondria. When Jurkat cells were induced to undergo apoptosis by Fas receptor ligation, cytochrome c was released from mitochondria, an event that was prevented by the caspase inhibitor, zVAD-fmk (zVal-Ala-Asp-CH2F). Purified caspase-8 triggered rapid cytochrome c release from isolated mitochondria in vitro. The effect was indirect, as the presence of cytosol was required, suggesting that caspase-8 cleaves and activates a cytosolic substrate, which in turn is able to induce cytochrome c release from mitochondria. The cytochrome c releasing activity was not blocked by caspase inhibition, but was antagonized by Bcl-2 or Bcl-xL. Caspase-8 and caspase-3 cleaved Bid, a proapoptotic Bcl-2 family member, which gains cytochrome c releasing activity in response to caspase cleavage. However, caspase-6 and caspase-7 did not cleave Bid, although they initiated cytochrome c release from mitochondria in the presence of cytosol. Thus, effector caspases may cleave and activate another cytosolic substrate (other than Bid), which then promotes cytochrome c release from mitochondria. Mitochondria significantly amplified the caspase-8 initiated DEVD-specific cleavage activity. Our data suggest that cytochrome c release, initiated by the action of caspases on a cytosolic substrates, may act to amplify a caspase cascade during apoptosis.     

Yeast two-hybrid screening using constitutive-active caspase-7 as bait in the identification of PA28gamma as an effector caspase substrate

Caspase-3 and -7 represent executioner/effector caspases that directly cause apoptotic morphological changes by cleaving various death substrates. The substrates for caspases generally interact with active caspases, but not with inactive zymogens of caspase or procaspases. Here, to isolate proteins that interact with caspase-7, we established a yeast two-hybrid screening system using reversed-caspase-7, a constitutive active mutant of caspase-7 as a bait plasmid. Screening of an adult brain cDNA library led to isolation of proteasome activator 28 subunit, PA28gamma. In vitro translates of PA28gamma were cleaved by both recombinant caspase-3 and -7. Mutagenesis of potential cleavage site DGLD80 to EGLE80 completely abolished caspase-mediated cleavage. Moreover, endogenous PA28gamma was cleaved during not only Fas-induced apoptosis of HeLa cells, but also cisplatin-induced cell death of MCF7 cells, which are devoid of caspase-3. These findings indicate that PA28gamma is an endogenous substrate for caspase-3 and -7 and that yeast two-hybrid screening using reversed-caspase is a novel and useful approach to clone substrates for effector caspases.     

Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation.

Caspase-3 initiates apoptotic DNA fragmentation by proteolytically inactivating DFF45 (DNA fragmentation factor-45)/ICAD (inhibitor of caspase-activated DNase), which releases active DFF40/CAD (caspase-activated DNase), the inhibitor's associated endonuclease. Here, we examined whether other apoptotic proteinases initiated DNA fragmentation via DFF45/ICAD inactivation. In a cell-free assay, caspases-3, -6, -7, -8, and granzyme B initiated benzoyloxycarbonyl-Asp-Glu-Val-Asp (DEVD) cleaving caspase activity, DFF45/ICAD inactivation, and DNA fragmentation, but calpain and cathepsin D failed to initiate these events. Strikingly, only the DEVD cleaving caspases, caspase-3 and caspase-7, inactivated DFF45/ICAD and promoted DNA fragmentation in an in vitro DFF40/CAD assay, suggesting that granzyme B, caspase-6, and caspase-8 promote DFF45/ICAD inactivation and DNA fragmentation indirectly by activating caspase-3 and/or caspase-7. In vitro, however, caspase-3 inactivated DFF45/ICAD and promoted DNA fragmentation more effectively than caspase-7 and endogenous levels of caspase-7 failed to inactivate DFF45/ICAD in caspase-3 null MCF7 cells and extracts. Together, these data suggest that caspase-3 is the primary inactivator of DFF45/ICAD and therefore the primary activator of apoptotic DNA fragmentation.     

ER stress induces caspase-8 activation,stimulating cytochrome c release and caspase-9 activation

Excess ER stress induces caspase-12 activation and/or cytochrome c release, causing caspase-9 activation. Little is known about their relationship during ER stress-mediated cell death. Upon ER stress, P19 embryonal carcinoma (EC) cells showed activation of various caspases, including caspase-3, caspase-8, caspase-9, and caspase-12, and extensive DNA fragmentation. We examined the relationship between ER stress-mediated cytochrome c/caspase-9 and caspase-12 activation by using caspase-9- and caspase-8-deficient mouse embryonic fibroblasts and a P19 EC cell clone [P19-36/12 (-) cells] lacking expression of caspase-12. Caspase-9 and caspase-8 deficiency inhibited and delayed the onset of DNA fragmentation but did not inhibit caspase-12 processing induced by ER stress. P19-36/12 (-) cells underwent apoptosis upon ER stress, with cytochrome c release and caspase-8 and caspase-9 activation. The dominant negative form of FADD and z-VAD-fmk inhibited caspase-8, caspase-9, Bid processing, cytochrome c release, and DNA fragmentation induced by ER stress, suggesting that caspase-8 and caspase-9 are the main caspases involved in ER stress-mediated apoptosis of P19-36/12 (-) cells. Caspase-8 deficiency also inhibited the cytochrome c release induced by ER stress. Thus, in parallel with the caspase-12 activation, ER stress triggers caspase-8 activation, resulting in cytochrome c/caspase-9 activation via Bid processing.     

Caspases in T-cell receptor-induced thymocyte apoptosis.

Apoptosis eliminates inappropriate or autoreactive T lymphocytes during thymic development. Intracellular mediators involved in T-cell receptor (TCR)-mediated apoptosis in developing thymocytes during negative selection are therefore of great interest. Caspases, cysteine proteases that mediate mature T-cell apoptosis, have been implicated in thymocyte cell death, but their regulation is not understood. We examined caspase activities in distinct thymocyte subpopulations that represent different stages of T-cell development. We found caspase activity in CD4+CD8+ double positive (DP) thymocytes, where selection involving apoptosis occurs. Earlier and later thymocyte stages exhibited no caspase activity. Only certain caspases, such as caspase-3 and caspase-8-like proteases, but not caspase-1, are active in DP thymocytes in vivo and can be activated when DP thymocytes are induced to undergo apoptosis in vitro by TCR-crosslinking. Thus, specific caspases appear to be developmentally regulated in thymocytes.     

Caspase Activation Accompanying Cytochrome c Release from Mitochondria Is Possibly Involved in Nitric Oxide-Induced Neuronal Apoptosis in SH-SY5Y Cells

Abstract : It is well known that caspases are produced as proforms, which are proteolytically cleaved and activated during apoptosis or programmed cell death. We report here that caspases are activated during apoptosis by treatment with NOC18, a nitric oxide (NO) donor. Our present experiments have examined the way in which NO induces neuronal cell death, using a new type of NO donor that spontaneously releases only NO without enzymatic metabolism. NOC18 induced apoptosis in human neuroblastoma SH-SY5Y cells in a concentration-and time-dependent manner as estimated by DNA fragmentation assay, FACScan analysis, and nuclear morphology. Oxyhemoglobin, an NO trapper, suppressed NOC18-triggered DNA fragmentation, indicating that NO from NOC18 is a real activator in this study. Upon the induction of apoptosis, an increase in caspase-3-like protease activity, but not caspase-1, was observed. Procaspase-2 protein, an inactive form of caspase-2, decreased dramatically. In addition, NOC18 also resulted in poly (ADP-ribose) polymerase (PARP) cleavage, yielding an 85-kDa fragment typical of caspase activity. Oxyhemoglobin blocked the decrease of procaspase-2 and the cleavage of PARP by NOC18 in a concentration-dependent manner. Moreover, NO elicited the release of cytochrome c into the cytosol during apoptosis. These results suggest that both stimulation of caspase activity and cytochrome c release are partly involved in NO-induced neuronal apoptosis.