|
|
|
|
|
|
|
||
Articles |
Department of Viral Oncology, Institute for Virus Research, Kyoto University, Kyoto 606, Japan; and the College of Medical Technology, Kyoto University, Kyoto 606, Japan
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Fas/APO-1/CD95 is a cell-surface receptor essential for the regulation of the immune system (1), especially for the termination of T cell–mediated responses, the maintenance of immune privilege (2), and the prevention of autoantibody production (3). Evidence is accumulating that a family of cysteine proteases, named caspases (4), play critical roles in Fas-induced apoptotic cell death. The inhibition of caspases by cowpox serpin CrmA or by synthetic peptide inhibitors such as zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp(OMe)- fluoromethylketone) and zDEVD-fmk (benzyloxycarbonyl-Asp-Glu-Val-Asp(OMe)-fluoromethylketone), allows Fas-stimulated cells to survive (5) and proliferate (6). Cross-linking of the Fas receptor with Fas ligand (7) or with anti-Fas mAb (8) recruits the death-inducing signaling complex (DISC)1 consisting of FADD/MORT1 and pro–caspase-8 (MACH/ FLICE/Mch5) (9, 10). Pro–caspase-8 recruited to the DISC is activated and released into the cytosol (11). The active caspase-8 activates multiple caspases (12–14), which elicit downstream biochemical changes such as mitochondrial permeability transition (PT; 15), DNA fragmentation (16), loss of nuclear lamina (17), and exposure of phosphatidylserine (PS) on the cell surface (18). Together, these changes culminate in the process of apoptosis (19). However, the activation of caspases downstream of caspase-8 is a poorly understood process. Nor is it clear which caspase(s) contributes to each apoptotic biochemical or morphological change.
Each caspase family protease is present in nonapoptotic cells as an inactive proenzyme. The enzyme becomes active when the precursor is cleaved into a large subunit with a molecular mass of
Affinity labeling techniques have been developed taking advantage of the ability of active caspases, not their inactive precursors, to bind their substrates. Derivatized peptides mimicking substrates for caspases enter the substrate-binding pockets of active caspases and irreversibly bind to the active site cysteine in the large subunits, allowing their detection on immunoblots (17, 28–30) or on histological sections (31). Application of this technique, combined with inhibitor studies (14), identified multiple active caspases including caspase-3–p20, caspase-3–p17, caspase-6 (Mch2), caspase-7 (Mch3/ ICE-LAP3/CMH-1), and caspase-8 in Fas-stimulated human Jurkat T cells. Stepwise appearances of active caspases (14) were highly suggestive of a cascade of caspase activation. However, a sufficiently detailed dissection of the activation pathway would have required inhibitors with high specificity for individual caspases, and the available caspase inhibitors such as DEVD-CHO had too broad of a spectrum for this purpose. Although CrmA is rather selective for caspase-1, -4, -5, and -8 (32–34), the inhibition of caspase-8 by CrmA completely suppresses subsequent activation of other caspases (35). This has made further analysis of downstream events difficult.
Here, we demonstrated that novel tetrapeptide inhibitors, VEID-CHO (acetyl-Val-Ileu-Asp-aldehyde) and DMQD-CHO (acetyl-Asp-Met-Gln-Asp-aldehyde), can work as specific inhibitors of caspase-6 and -3, respectively. These reagents were used to analyze the caspase activation induced by Fas ligation in Jurkat cells with that elicited by caspase-8 in cell-free Jurkat extracts. In each case, caspase-6 is activated downstream of caspase-3, forming a protease cascade. Moreover, we identified the caspases upstream of each caspase-dependent event in Fas-stimulated Jurkat cells.
Inhibition of Caspases in Fas-stimulated Cells and in a Cell-free Reaction Triggered by Caspase-8.
Single-cell Analysis of Intracellular Caspase Activity in Fas-stimulated Jurkat Cells.
Immunoblotting for the Cleavage of Caspase Substrates.
Flow Cytometric Analysis of DNA Fragmentation, Mitochondrial Membrane Potential, and Cell-surface Exposure of PS.
20 kD and a small subunit with a molecular mass of 10 kD, and forms a tetramer consisting of two large and two small subunits (20). Most well-known activation sites are immediately after aspartic acid (D) residues of pro-caspases (21), and the unique characteristic of caspases is that they cleave after aspartic acids within their substrates (22). This suggests that caspases are activated by autocatalysis or by mutual processing (23). Different cascade pathways for the activation of caspases have been proposed, based on the ability of certain recombinant caspases to cleave and activate a limited spectrum of recombinant pro-caspases in vitro and on the cleavage of endogenous pro-caspases within cells in which a caspase(s) is exogenously overexpressed (12, 24–26). However, these studies did not identify the cascades that are induced in cells in response to death-inductive stimuli. A study using YVAD-CHO, which inhibits caspase-1 (IL-1β converting enzyme; ICE), and DEVD-CHO, which inhibits caspase-3 (CPP32/Yama/ apopain), indicated that there is a protease cascade in which caspase-1–like activity is upstream of caspase-3–like activity in Fas-mediated apoptosis of mouse W4 cells (27). However, the caspases involved in this cascade, especially the identity of the caspase-1–like protease(s), remain unclear.
Materials and Methods
Top
Abstract
Materials and Methods
Results
Discussion
References
Reagents.
Rabbit polyclonal antiserum against the large subunit of caspase-7 (36) was provided by Gerald M. Cohen (University of Leicester, Leicester, UK). DMQD-CHO (14), VEID-CHO (Peptide Institute, Osaka, Japan), and zVAD-fmk (Enzyme Systems, Dublin, CA) were dissolved at 10 mM in DMSO and stored at –80°C. DiOC6(3) (3, 3'-dihexyloxacarbocyanine iodide; Molecular Probes Inc., Eugene, OR) was dissolved at 0.5 mM in DMSO and stored at –20°C. Propidium iodide (Calbiochem Corp., La Jolla, CA) was dissolved in PBS at 100 µg/ml and stored at 4°C.
Jurkat cells were pretreated with VEID-CHO or DMQD-CHO for 1 h at 37°C before stimulation with anti-Fas mAb (CH-11, 100 ng/ml; reference 8). Preparation of cytoplasmic extracts and the affinity labeling of active caspases using YV(bio)KD-aomk (provided by Nancy A. Thornberry, Merck, Rahway, NJ; reference 28) were performed as previously described (14). Cytoplasmic extracts from logarithmically growing Jurkat cells were preincubated with VEID-CHO or DMQD-CHO for 1 h at 37°C before the addition of purified recombinant active caspase-8 to trigger the stepwise activation of caspases (14).
Jurkat cells (2.5 x 105) treated with anti-Fas mAb were gently centrifuged in a microfuge tube. The cell pellet was resuspended with 50 µl of 10 µM PhiPhiLux-G2D2 substrate solution (OncoImmunin, College Park, MD) in RPMI-1640 supplemented with 10% FCS. After incubation for 1 h at 37°C avoiding direct light, the sample was diluted with 0.5 ml of ice-cold flow cytometry dilution buffer (OncoImmunin) and filtered through a nylon mesh to remove cell aggregates and/or debris. Flow cytometric analysis was performed within 90 min of the end of the incubation period using FACScan® flow cytometer and LYSIS II software (Becton Dickinson, Mountain View, CA).
Nuclei isolated from HeLa cells as described (37) were incubated with purified recombinant caspases, with Escherichia coli lysates containing recombinant caspases (14), or with the extract from preapoptotic chicken DU249 cells (S/M extract; reference 37). Whole cell extracts from Jurkat cells were obtained by adding SDS-PAGE sample buffer (50 mM Tris-HCl [pH 6.8], 15% sucrose, 3% SDS, and 0.01% bromophenol blue) to cell pellets and boiling for 3 min. Proteins were resolved in SDS-PAGE gels, transferred to nitrocellulose membranes (Hybond ECL, Amersham Corp., Arlington Heights, IL), and stained with rabbit polyclonal anti–nuclear mitotic apparatus protein (NuMA) antiserum (r240-C; reference 38), anti–poly (ADP-ribose) polymerase (PARP) mAb (C-2-10; BIOMOL, Plymouth Meeting, PA), or rabbit polyclonal anti-ste20-subdomain VI Ab (Upstate Biotechnology Inc., Lake Placid, NY). Signals were visualized by enhanced chemiluminescence reagents (Amersham Corp.) according to the manufacturer's instructions.
For the analysis of DNA fragmentation, Jurkat cells (106) were permeabilized by fixation with 70% ethanol for 2 h on ice, washed once with PBS, treated with 10 µg/ml RNase A in PBS for 30 min at 37°C, and incubated with 50 µg/ml propidium iodide for 30 min on ice protected from light. For simultaneous recording of mitochondrial PT and PS externalization, Jurkat cells (5 x 105) were incubated with 40 nM DiOC6 (3) (Molecular Probes) together with 1.5 µg/ml PE-conjugated annexin V (R&D Sys. Inc., Minneapolis, MN) in 10 mM Hepes/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2 for 15 min at room temperature. Stained cells were filtered through a nylon mesh and immediately subjected to flow cytometry.
Results
Top
Abstract
Materials and Methods
Results
Discussion
References
Affinity Labeling of Active Caspases in Fas-stimulated Jurkat Cells.
YV(bio)KD-aomk (28), an affinity-labeling reagent for active caspases (14), recognized four major polypeptides in Fas-stimulated Jurkat cells, designated F22, F20, F19, and F17 according to their apparent molecular mass in kilodaltons (Fig. 1 A, lane 1). Previous studies revealed that these correspond to caspase-7, caspase-3-p20, caspase-6, and caspase-3-p17, respectively (14, 30). Using rabbit antiserum raised against caspase-7 (36), we have confirmed that F22 corresponds to caspase-7 (Fig. 1 B, lane 1, and data not shown).
|
|
Analysis of Caspase Cascade by Selective Inhibition of Caspase-3 with DMQD-CHO.
DMQD-CHO was synthesized based on the cleavage site within protein kinase C 
(PKC). Although PKC is cleaved by caspase-3, it is not a substrate for caspase-1, -2, -4, -5, -6, or -7 (42). Our previous study showed that F20/caspase-3-p20 is inhibited at lower concentrations than either F22/caspase-7 or F19/caspase-6 (14). Caspase-8 is also relatively insensitive to DMQD-CHO (data not shown). First, we investigated the effects of DMQD-CHO on the cell-free activation of caspases in Jurkat cytoplasmic extracts triggered by recombinant caspase-8 (14). YV(bio)KD-aomk labeling of F20/caspase-3-p20 was inhibited by DMQD-CHO at 1–2 µM (Fig. 2 A, lanes 4 and 5), the concentrations previously shown to block the activity of F20/caspase-3-p20 (14). The labeling of F17/ caspase-3-p17 was also suppressed, most likely because the cleavage of the prodomain of caspase-3-p20, which generates caspase-3-p17, is mediated by autocatalysis (25, 43). Interestingly, although the activity of F19/caspase-6 is insensitive to DMQD-CHO even at 100 µM (14), the appearance of F19/caspase-6 was prevented by the preincubation with 1–2 µM DMQD-CHO before the addition of caspase-8 (Fig. 2 A, lanes 4 and 5). This indicated that the activation of F19/caspase-6 in response to caspase-8 is dependent on the activity of F20/caspase-3-p20. This finding is supported by a previous report in which efficient activation of caspase-6 by caspase-8 in a cell-free system requires the presence of cytoplasmic extracts (13). This suggests that caspase-6 is activated indirectly via another protease(s) present in the extracts. In contrast, the labeling of F22/caspase-7 was unhindered up to 5 µM DMQD-CHO (lane 6). This indicated that caspase-7 is activated independent of caspase-3 activity. The loss of all labeling at 10 µM (lane 7) is due to the inhibition of caspase-8 (data not shown).
|
Are caspases organized in a similar cascade within Fas-stimulated Jurkat cells? Preincubation of Jurkat cells with 10–100 µM DMQD-CHO before Fas receptor cross-linking suppressed the labeling of F20/caspase-3-p20 and F17/ caspase-3-p17. Concomitant with the suppression of caspase-3 activity, the appearance of F19/caspase-6 was also blocked (Fig. 2 C, lanes 3 and 4). In contrast, the labeling of F22 persisted up to 100 µM DMQD-CHO (Fig. 2 C, lane 4). Reprobing of the same blot with anti–caspase-7 Ab confirmed that F22 in the presence of 1–100 µM DMQD-CHO corresponds to the large subunit of caspase-7 (data not shown). As the concentration of DMQD-CHO required to block the activity of caspase-6 is 10-fold higher than caspase-7 (14), the loss of caspase-6 labeling with preserved caspase-7 activity indicated that the suppression of caspase-3-p20 activity resulted in the loss of caspase-6 activation. Time course studies (Fig. 2 D) demonstrated that active F22/caspase-7 appeared at the same time point irrespective of the presence of DMQD-CHO. This result showed that activation of caspase-7 was uninterrupted when caspase-3 activity was blocked with 100 µM DMQD-CHO.
Demonstration of Caspase Activity in Intact Fas-stimulated Jurkat Cells Pretreated with DMQD-CHO.
We were concerned about the possibility that caspase-7 was inhibited by DMQD-CHO in Jurkat cells, whereas it was reactivated by reversible dissociation from the inhibitor when the cells were disrupted to prepare cytoplasmic extracts. Thus, we measured caspase activity in intact Fas-stimulated cells by flow cytometry, using PhiPhiLux-G2D2. This cell-permeable fluorescent substrate for caspases emits increased fluorescence when it is proteolyzed within its PARP cleavage site sequence (GDEVDGID). Since caspase-3 and -7 appear to be primarily responsible for PARP cleavage during apoptosis (21), the increase in fluorescence should mainly reflect the activities of caspase-3 and -7. Fig. 3 A depicts time course studies displaying the forward light scatter (FSC), an estimate of cell size, and the PhiPhiLux-G2D2 fluorescence of Jurkat cells (FL2). After Fas ligation, a gradual augmentation of the PhiPhiLux-G2D2 fluorescence was noted along with a decrease in FSC, suggesting the activation of caspases concomitant with the shrinkage of cytoplasm. The increase in fluorescence correlated well with the kinetics of caspase activation measured by the ability of Jurkat cytoplasmic extracts to cleave DEVD-MCA (14). The enhanced PhiPhiLux-G2D2 fluorescence and the decrease in FSC was not observed on incubation of the cells without anti-Fas mAb (data not shown). The increase in fluorescence was abolished by the presence of 100 µM zVAD-fmk (Fig. 3 A), a broad-spectrum caspase inhibitor that blocks the activation of PARP-cleaving caspases (44), supporting the notion that caspases are responsible for cleavage of PhiPhiLux-G2D2. Lower fluorescence was observed in the population of cells with the lowest FSC, possibly due to the loss of the fluorescent molecules by budding out of apoptotic bodies. In the presence of DMQD-CHO at 100 µM, only minimal suppression of protease activity was noted, with the PhiPhiLux-G2D2 fluorescence significantly enhanced (Fig. 3 B, DMQD 100 µM) compared with untreated cells (Fig. 3 B, control). This was consistent with the persistence of caspase-7 activity demonstrated by affinity labeling (Fig. 2 C, lane 4). As expected, incubation with 100 µM DMQD-CHO alone without Fas simulation did not induce the increase in PhiPhiLux-G2D2 fluorescence or cell shrinkage (data not shown).
|
|
190 and 180 kD. Time course studies revealed that the 190-kD fragment was the first to be detected, then disappeared as the 180-kD fragment gradually appeared (data not shown). This suggested that the 180 kD fragment was derived from proteolysis of the 190-kD fragment. Caspase-6 also yielded a fragment with Mr of 160 kD in addition to the 190- and 180-kD fragments (Fig. 5 A, lane 6). These fragments comigrated with those detected in the cell-free system composed of HeLa nuclei and S/M extracts (Fig. 5 A, lane 7). Jurkat cells undergoing apoptosis in response to 1 µM staurosporine (14) showed two distinct fragments of NuMA with Mr of 180 and 160 kD (Fig. 5 A, lane 8) comigrating with those of HeLa nuclear NuMA generated by recombinant caspases or by caspases in S/M extracts. Similar results were obtained using purified recombinant human NuMA and recombinant caspase-3, -4, -6, -7, -8, or S/M extracts (data not shown). Thus, these caspases can cleave NuMA protein directly.
|
180 and 160 kD (Fig. 5 B, lane 1). In the presence of 1–10 µM VEID-CHO, the production of the 160-kD fragment in Fas-stimulated Jurkat cells was suppressed (Fig. 5 B, lanes 7 and 8), in parallel with the loss of caspase-6 activity (Fig. 1 A, lanes 4 and 5). This is consistent with the unique ability of recombinant caspase-6 to generate the 160-kD fragment, and indicates that caspase-6 is responsible for the cleavage producing this fragment in Fas-stimulated Jurkat cells. In contrast, the proteolysis yielding the 180-kD fragments was not affected at 1 µM (Fig. 5 B, lane 7). Although recombinant caspase-6 is able to produce the 190- and 180-kD fragments in vitro, it is not the major protease responsible for the production of those fragments in apoptotic Jurkat cells. The moderate decrease in the 180-kD fragment at 10 µM VEID-CHO (Fig. 5 B, lane 8) was considered to reflect the inhibition of the activities of caspase-3-p17 as well as caspase-6 at this concentration (Fig. 1 A, lane 5). Interestingly, 100 µM DMQD-CHO blocked the generation of the 180-kD fragment as well as the 160-kD fragment. These results indicated that caspase-3 is responsible for the production of the 180-kD fragment in Fas-stimulated Jurkat cells. Although caspase-7 is able to cleave HeLa nuclear and recombinant NuMA in vitro (Fig. 5 A, lane 4, and data not shown), the presence of caspase-7 activity alone was not sufficient for the cleavage of NuMA in Fas-stimulated Jurkat cells. Thus, there is a discrepancy between the cell-free and cellular results. This discrepancy may be explained by the unique intracellular localization of caspase-7 within intact cells. Caspase-7 has been detected solely in the cytoplasm of Jurkat cells (48), whereas caspase-3 can be detected in the nuclei of some cell types (49) and of regressing, apoptotic neuroblastoma cells (50). In cell-free systems, nuclei may be permeabilized to some extent, allowing free entry of proteins normally inaccessible to the intranuclear compartment. To test this possibility, we examined whether the cleavage of another nuclear substrate, PARP, is mediated solely by caspase-3 in Jurkat cells. In contrast to NuMA, 100 µM DMQD-CHO only weakly inhibited the PARP cleavage (Fig. 5 C, lane 4). Suppression of caspase-6 activity with 1 µM VEID-CHO did not affect the PARP cleavage (Fig. 5 C, lane 6). Partial blockade of the cleavage with 10 µM VEID-CHO (Fig. 5 C, lane 7) was likely to reflect the loss of caspase-3-p17 activity (Fig. 1 A, lane 5). PARP cleavage was markedly suppressed by 100 µM VEID-CHO (Fig. 5 C, lane 8). These indicated that caspase-3 is only partially responsible for PARP cleavage within Fas-stimulated Jurkat cells. Thus, another caspase(s), most likely caspase-7, cleaves PARP in the cells. This suggests that caspase-7 can enter the nucleus and cleave its nuclear substrate(s). The basis for the difference between caspase-3 and -7 in the ability to cleave NuMA in Jurkat cells remained unclear.
Role of Caspase-3 and -6 in Nuclear Apoptotic Changes.
The inhibition of caspase-3 plus -6 by 100 µM DMQD-CHO completely blocked the nuclear morphological changes as revealed by staining of chromatin structures with 4',6-diamidino-2-phenylindole (DAPI; Fig. 6 h). In contrast, the inhibition of caspase-6 alone with 10 µM VEID-CHO resulted in the blockade of apoptotic morphology halfway after the nuclear chromatin condensation into the nuclear periphery ("rim collapse"; Fig. 6 d). These indicate that caspase-3 is essential for the early morphological changes of nuclei up to the rim collapse, whereas caspase-6 plays an essential role in the late morphological changes such as the shrinkage and fragmentation of apoptotic nuclei. This is consistent with previous results in a cell-free system that a lamin-cleaving caspase is responsible for the late nuclear events, whereas a different caspase mediates the initiation of nuclear morphological changes (51–53).
|
|
|
Identification of Caspases Upstream of Extranuclear Morphological and Biochemical Changes.
The shrinkage of cytoplasm in Fas-stimulated Jurkat cells proceeded to a significant degree despite the blockade of both caspase-3 and -6 by 100 µM DMQD-CHO (Figs. 3 B and 7 B). Although the selective blockade of caspase-6 with 10 µM VEID-CHO had no effect on cell size (Fig. 7 B), the broad-spectrum blockade of all caspases at 100 µM VEID-CHO markedly suppressed the decrease in cell size (Fig. 7 B). The shapes of cells observed by phase contrast microscopy were consistent with the flow cytometric data. Neither 100 µM DMQD-CHO (Fig. 8 h) nor 10 µM VEID-CHO (Fig. 8 d) affected the Fas-induced shrinkage of Jurkat cells. In contrast, cytoplasmic shrinkage was completely blocked with 100 µM VEID-CHO (Fig. 8 e). Taken together, a caspase(s) other than caspase-3 or -6 plays a critical role(s) in the Fas-induced shrinkage of cytoplasm.
To delineate further the involvement of each caspase in extranuclear processes, we examined apoptotic biochemical changes in mitochondria and the plasma membrane. Breakdown of the inner mitochondrial membrane potential (mitochondrial PT) is one of the earliest biochemical changes in cells undergoing apoptosis (55, 56). Although recent reports indicate that Fas-induced PT is dependent on the activity of caspases (5, 15, 57), it remained to be determined which caspase(s) is upstream of PT in the cells. The loss of plasma membrane asymmetry, resulting in the surface exposure of PS (58) and phosphatidylethanolamine (59), is another characteristic apoptotic change. Fas-induced PS externalization depends on the activity of caspases (18). We measured PT and PS externalization by double staining with DiOC6(3), the fluorescent probe that is sequestered to mitochondria depending on the membrane potential (60), and PE-conjugated annexin V, a natural protein that binds PS exposed to the cell surface with high affinity (61). As shown in Fig. 9 A, a cell population with decreased DiOC6(3) uptake and increased annexin V binding was first noted at 1 h after Fas ligation. This population with collapsed mitochondrial membrane potential and with externalized PS increased over time. Neither PT nor PS externalization were affected by 10 µM VEID-CHO (Fig. 9 B). The blockade of all caspases with 100 µM VEID-CHO markedly inhibited both processes (Fig. 9 B), confirming previous reports that those events are downstream of caspases. DMQD-CHO at 100 µM suppressed the increase in annexin V binding (Fig. 9 B). Thus, the activity of caspase-3 is essential for PS externalization. In contrast, PT was not affected by 100 µM DMQD-CHO, indicating that a caspase(s) other than caspase-3 or -6 is involved in the disruption of mitochondrial membrane potential in Fas-stimulated Jurkat cells.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The ability of caspase-8 to induce stepwise activation of caspases in organelle-free cytoplasmic extracts of Jurkat cells (14) suggests that mitochondria are dispensable for the organization of this protease cascade in Jurkat cells. However, this does not rule out the possibility that caspase-8 indirectly activates caspase-3 by inducing mitochondrial release of cytochrome c (62, 63) and/or AIF (57) in other cell types. In cells with more cytoplasm and a lower concentration of pro-caspase-3, caspase-8 activated in the DISC on the plasma membrane and released into the cytoplasm might not process cytoplasmic pro-caspase-3 efficiently. The sequestration of caspase-8 to the mitochondria (64) and the subsequent release of cytochrome c and AIF could well facilitate the activation of caspase-3. This pathway should be particularly active in cells rich in mitochondria. The existence of both direct and indirect pathways for the activation of caspase-3 by caspase-8 would explain the differences among cell types in the protective effect of Bcl-2 and Bcl-xL on Fas-induced cell death (15, 35). In cells that predominantly use the indirect pathway for caspase-3 activation, Bcl-2 family proteins may inhibit Fas-induced apoptosis by blocking the release of cytochrome c and/or AIF from mitochondria.
It seems unlikely that caspase-8 activates caspase-3 via other caspases. Affinity labeling displayed three other caspases, caspase-7, caspase-6, and F25, in Fas-stimulated Jurkat cells (14). Inhibitor studies with VEID-CHO showed that caspase-6 activity is not required for the activation of caspase-3. However, recent evidence suggests that different caspase cascades may be used depending on cell types and death-inductive stimuli (65). In some cell death events, caspase-6 may act upstream of caspase-3 (24, 41). The appearance of F25 is a delayed event, much later than caspase-3, -7, and -6 (14). Therefore, it seems unlikely that F25 is upstream of other caspases. Previous studies indicated that recombinant caspase-7 cannot process recombinant pro-caspase-3 (66). Indeed, the addition of recombinant active caspase-7 to the extracts from normally growing Jurkat cells failed to induce F20/ caspase-3-p20 (data not shown). Overall, our findings strongly suggested that there is a direct two-step protease cascade in Fas-stimulated Jurkat cells, in which caspase-8 activated via FADD/MORT1 cleaves pro-caspase-3, and the activated caspase-3 in turn activates caspase-6 by directly cleaving pro-caspases-6 (Fig. 10).
|
Detailed analyses of Fas-induced signaling using VEID-CHO and DMQD-CHO provided evidence that the protease cascade bifurcates into a caspase-7 arm and a caspase-3 arm, each inducing distinct downstream intracellular events (Fig. 10). There is a point of ramification in the Fas signaling pathway at the level of the caspase cascade.
Caspase-3 Arm.
The caspase-3 arm seems to play major roles in the execution of nuclear apoptosis. Caspase-3 activates caspase-6, which in turn plays an essential role in the induction of nuclear shrinkage and nuclear fragmentation. Cleavage of the nuclear structural protein NuMA is mediated exclusively by the caspases of this branch. DNA fragmentation, a hallmark of nuclear apoptosis (67), is downstream of caspase-3. This is consistent with the result in a cell-free system showing that caspase-3 is a direct activator of DNA fragmentation factor (DFF; reference 16). DFF can induce DNA ladder in isolated nuclei only after its 45-kD subunit is cleaved by caspase-3. Our present data support the idea that caspase-3 mediates DNA fragmentation by cleaving DFF in Fas-stimulated Jurkat cells.
Microscopic analysis of DAPI-stained nuclei indicated that caspase-3 has a role in the initiation of chromatin condensation. PKC is cleaved and activated by caspase-3, not by caspase-6 or -7 (42). By undetermined mechanisms, introduction of cleaved active PKC fragment to HeLa cells induces nuclear apoptotic changes, including chromatin condensation (42). This may underlie the ability of caspase-3 to initiate the nuclear apoptotic morphological changes.
Although caspase-3 plays major roles in nuclear apoptotic events, it is also involved in extranuclear processes. PS exposed on the cell surface is recognized by macrophages and triggers the engulfment of apoptotic cells by phagocytes (58). Our results indicated that this PS externalization in response to Fas ligation is downstream of caspase-3. The molecular mechanism by which caspase-3 mediates PS externalization remains to be determined. It has been suggested that PS exposure results from the inactivation of aminophospholipid translocase and the enhanced activity of scramblase (68). In the recently reported amino acid sequence of scramblase (69), however, no canonical consensus sequence for caspase-3 cleavage (22) was identified. We found that caspase-3 is a principal mediator of the apoptotic body formation. This may reflect the fact that PAK2 cleavage in Fas-stimulated Jurkat cells is mainly catalyzed by caspase-3. Alternatively, the apoptotic body formation may be coupled to nuclear events, as suggested by the exclusion of Ro, La, and small nuclear ribonucleoproteins (snRNPs) from the nucleoplasms and the clustering of these ribonucleoproteins in apoptotic bodies (70).
Caspase-7 Arm.
The caspase-7 arm is involved in the Fas-induced signaling in a manner distinct from caspase-3 and -6. Our results suggested that caspase-7 catalyzes, together with caspase-3, the cleavage of a nuclear substrate, PARP. This result accords well with the analysis of caspase-3 knockout mice showing that the PARP cleavage in apoptotic thymocytes is seemingly unaffected by the loss of caspase-3 (65). Thus, caspase-7 can compensate for some of the apoptotic biochemical events mediated by caspase-3.
Our observations suggest that caspase-7 also has a role(s) in the Fas-induced signaling that are not redundant with caspase-3. Apoptotic shrinkage of cytoplasm (19) persisted in the presence of 100 µM DMQD-CHO, which blocked caspase-3 and -6. A recent report described a similar partial blockade of apoptotic events in KB epidermal carcinoma cells. zDEVD-FK at 50 µM abolished Fas-induced chromatin condensation and apoptotic body formation, whereas surface blebbing, cell shrinkage, the disruption of actin network, and the activation of stress-activated MAP kinases, c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38, remained uninterrupted (71). In contrast, the blockade of all caspases with 100 µM VEID-CHO abolished the cell shrinkage. Caspase-7, active in the presence of DMQD-CHO, may thus mediate cell shrinkage. Although caspase-8 might mediate cell shrinkage, active caspase-8 was not detected in staurosporine-induced apoptosis of Jurkat cells (14) in which cytoplasmic shrinkage is prominent. Further, although caspase-3 and -7 have closely related structures (66) and almost identical preferences for tetrapeptide substrates (72, 73), caspase-7 may play a role(s) distinct from caspase-3. In this regard, it is of interest that caspase-3 and -7 have different optimal pH ranges (74). Further elucidation of cytoplasmic substrates specific for caspase-7 may clarify its role(s) in the cytoplasmic shrinkage induced by Fas ligation.
Can Caspase-7 Substitute for Caspase-3?
A controversial issue is whether caspase-7 can substitute for caspase-3. The substrate specificity of caspase-7 is similar to caspase-3 (72, 73), although caspase-7 cannot cleave some caspase-3 substrates such as PKC (42) and pro-caspase-6 (26). This study demonstrated that many biochemical changes of Fas-induced apoptosis in Jurkat cells are mediated solely by caspase-3 (Fig. 10). However, in knockout mice deficient in caspase-3, most apoptotic events are not interrupted, except for those in neuronal development (65). One attractive possibility is that caspase-3 and -7 compete for a common cofactor(s) to fulfill their intracellular functions. In Jurkat cells, caspase-3 inactivated by 100 µM DMQD-CHO may still compete with caspase-7 for the cofactor(s). In the absence of caspase-3 protein (65), however, caspase-7 may be free to use the cofactor(s) and thus to compensate for the loss of caspase-3 protein.
Mitochondrial PT.
Analysis of the mitochondrial transmembrane potential in Fas-stimulated cells showed that a caspase(s) other than caspase-3 or -6 is required for PT. Since the cell shrinkage is also mediated by a caspase(s) other than caspase-3 or -6, PT may be upstream of Fas-induced cytoplasmic shrinkage. It is of interest in this regard that the caspase-independent cell death induced by Bax, which may be a direct trigger of PT (75), is accompanied by PT and cell shrinkage (5). Because AIF is released on PT and activates DNase involved in internucleosomal DNA cleavage (76), it is surprising that DNA fragmentation was not observed despite the occurrence of PT in the presence of 100 µM DMQD-CHO. It is unclear whether the release of AIF is inhibited in the presence of DMQD-CHO, whether the activity of AIF is suppressed by the inhibitor, or whether the activity of AIF alone is not sufficient for the induction of DNA fragmentation.
We could not unequivocally determine which caspase is upstream of PT in Fas-stimulated Jurkat cells. However, it has been reported that caspase-8 transfected to 293T cells can form a complex with cotransfected Bcl-xL (64). If caspase-8 is recruited to mitochondria via complex formation with such Bcl-2 family proteins, it might act on mitochondrial substrates and disrupt the membrane potential. The development of an inhibitor(s) selective for caspase-7 should allow the identification of a caspase(s) responsible for such extranuclear events as PT and cytoplasmic shrinkage.
Cell-free versus Cellular Apoptosis.
A constant concern of in vitro studies is their applicability to intact cells. Although caspase-7 cleaves recombinant and HeLa nuclear NuMA in vitro, the endogenous NuMA of Jurkat cells remained uncleaved despite caspase-7 activation. Despite the ability of caspase-6 to generate the 180-kD fragment in vitro, only caspase-3 was responsible for this cleavage in Fas-stimulated Jurkat cells. Similarly, caspase-7 combined with extracts from nonapoptotic cells can induce DNA fragmentation in isolated nuclei, without activation of caspase-3 (24). However, a blockade of caspase-3 abolished DNA fragmentation within Fas-stimulated Jurkat cells despite persistent caspase-7 activity. In contrast, it is worth noting that the role of a lamin-cleaving protease(s), such as caspase-6, in the shrinkage and fragmentation of nuclei (Fig. 10) was first established in a cell-free system (51, 52). Cell-free systems have their own roles in the development of new tools and concepts, which should be explored at the level of cells and organisms (77).
Our approach exemplified the use of synthetic inhibitors that are relatively selective for each caspase. Recognition of substrates that are cleaved by one or a few caspases (17, 42) is a vital step in the design of such selective caspase inhibitors. The application of these reagents leads to effective approaches for dissecting the signaling pathways of apoptotic cell death. Inhibitors are being designed as the results of systematic screening of defined sequence variants of preferred substrates (72) and by use of the positional scanning synthetic combinatorial library (73). Detailed analysis of each cell death event using an array of inhibitors may identify targets for therapeutic interventions of the pathological processes resulting from excessive (78) or inappropriately suppressed (79) apoptosis.
Overall, the present study shows that the Fas-induced apoptotic cell death process is an integration of multiple, parallel biochemical events that occur downstream of the activation of particular caspases. The apparent heterogeneity in the biochemistry of apoptosis in different systems may be the result of the activation of different combinations of caspases, and consequently from their organization into various different protease cascades.
Submitted: 9 October 1997
Revised: 1 December 1997
, protein kinase C ; PS, phosphatidylserine; PT, permeability transition; VEID-CHO, acetyl-Val-Ileu-Asp-aldehyde; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone. We thank Emad S. Alnemri for the generous gift of cDNAs for caspases; Nancy A. Thornberry for providing YV(bio)KD-aomk; Gerald M. Cohen for anti–caspase-7 Ab; Charles H. Yang for anti-NuMA Ab; Alastair Mackay for critical review of the manuscript and creative suggestions; Yuri A. Lazebnik, Scott H. Kaufmann, William C. Earnshaw, Eisuke Nishida, Fumiko Toyoshima, and Katsumi Takada for helpful discussion and thoughtful suggestions; and Akira Komoriya, Kuniko Takano, Akinori Maeda, and Kouhei Yamashita for help in flow cytometric analysis.
A. Takahashi is a Research Resident of the Japanese Foundation of Aging and Health.
Address correspondence to Atsushi Takahashi, The First Division, Department of Internal Medicine, Kyoto University Hospital, 54 Shogoin Kawara-cho, Sakyo-ku, Kyoto 606, Japan. Phone: 81-75-751-4290; Fax: 81-75-751-4221; E-mail: atakahas{at}kuhp.kyoto-u.ac.jp
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
1 Nagata S. Apoptosis by death factor, Cell, 1997, 88, 355–365.[Medline]
2 Abbas AK. Die and let live: eliminating dangerous lymphocytes, Cell, 1996, 84, 655–657.[Medline]
3 Rathmell JC, Townsend SE, Xu JC, Flavell RA & Goodnow CC. Expansion or elimination of B cells in vivo: dual roles for CD40- and Fas (CD95)-ligands modulated by the B cell antigen receptor, Cell, 1996, 87, 319–329.[Medline]
4 Alnemri ES, Livingston DJ, Nicholson DW, Salvesen G, Thornberry NA, Wong WW & Yuan J. Human ICE/CED-3 protease nomenclature, Cell, 1996, 87, 171, .[Medline]
5 Xiang J, Chao DT & Korsmeyer SJ. BAX-induced cell death may not require interleukin 1β–converting enzyme– like proteases, Proc Natl Acad Sci USA, 1996, 93, 14559–14563.
6 Longthorne VL & Williams GT. Caspase activity is required for commitment to Fas-, mediated apoptosis, EMBO (Eur Mol Biol Organ) J, 1997, 16, 3805–3812.[Medline]
7 Nagata S & Goldstein P. The Fas death factor, Science, 1995, 267, 1449–1456.
8 Yonehara S, Ishii A & Yonehara M. A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor, J Exp Med, 1989, 169, 1747–1756.
9 Boldin MP, Goncharov TM, Goltsev YV & Wallach D. Involvement of MACH, a novel MORT1/ FADD-interacting protease in Fas/APO-1– and TNF receptor–induced cell death, Cell, 1996, 85, 803–815.[Medline]
10 Muzio M, Chinnaiyan AM, Kischkel FC, O'Rourke K, Shevchenko A, Ni J, Gentz R, Mann M, Krammer PH, Peter ME & Dixit VM. FLICE, a novel FADD-homologous ICE/CED-3–like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex, Cell, 1996, 85, 817–827.[Medline]
11 Medema JP, Scaffidi C, Kischkel FC, Shevchenko A, Mann M, Krammer PH & Peter ME. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC), EMBO (Eur Mol Biol Organ) J, 1997, 16, 2794–2804.[Medline]
12 Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Litwack G & Alnemri ES. Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases, Proc Natl Acad Sci USA, 1996, 93, 14486–14491.
13 Muzio M, Salvesen GS & Dixit VM. FLICE induced apoptosis in a cell-free system – cleavage of caspase zymogens, J Biol Chem, 1997, 272, 2952–2956.
14 Takahashi A, Hirata H, Yonehara S, Imai Y, Lee KK, Moyer RW, Turner PC, Mesner PW, Okazaki T, Sawai H et al.. Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8, Oncogene, 1997, 14, 2741–2752.[Medline]
15 Boise LH & Thompson CB. Bcl-x (L) can inhibit apoptosis in cells that have undergone Fas-induced protease activation, Proc Natl Acad Sci USA, 1997, 94, 3759–3764.
16 Liu XS, Zou H, Slaughter C & Wang XD. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis, Cell, 1997, 89, 175–184.[Medline]
17 Takahashi A, Alnemri ES, Lazebnik YA, Fernandes-Alnemri T, Litwack G, Moir RD, Goldman RD, Poirier GG, Kaufmann SH & Earnshaw WC. Cleavage of lamin A by Mch2
but not CPP32: multiple ICE-related proteases with distinct substrate recognition properties are active in apoptosis, Proc Natl Acad Sci USA, 1996, 93, 8395–8400.
18 Martin SJ, Finucane DM, Amarante-Mendes GP, O'Brien GA & Green DR. Phosphatidylserine externalization during CD95-induced apoptosis of cells and cytoplasts requires ICE/CED-3 protease activity, J Biol Chem, 1996, 271, 28753–28756.
19 Webb, S.J., D.J. Harrison, and A.H. Wyllie. 1997. Apoptosis: an overview of the process and its relevance in disease. In Apoptosis: Pharmacological Implications and Therapeutic Opportunities. S.H. Kaufmann, editor. Academic Press, San Diego. 1–34.
20 Rotonda J, Nicholson DW, Fazil KM, Gallant M, Gareau Y, Labelle M, Peterson EP, Rasper DM, Ruel R, Vaillancourt JP et al.. The three-dimensional structure of apopain/CPP32, a key mediator of apoptosis, Nat Struct Biol, 1996, 3, 619–625.[Medline]
21 Cohen GM. Caspases: the executioners of apoptosis, Biochem J, 1997, 326, 1–16.[Medline]
22 Nicholson DW & Thornberry NA. Caspases: killer proteases, TIBS (Trends Biochem Sci), 1997, 22, 299–306.[Medline]
23 Martin SJ & Green DR. Protease activation during apoptosis: death by a thousand cuts? , Cell, 1995, 82, 349–352.[Medline]
24 Orth K, O'Rourke K, Salvesen GS & Dixit VM. Molecular ordering of apoptotic mammalian CED-3/ ICE-like proteases, J Biol Chem, 1996, 271, 20977–20980.
25 Fernandes-Alnemri T, Armstrong RC, Krebs J, Srinivasula SM, Wang L, Bullrich F, Fritz LC, Trapani JA, Tomaselli K, Litwack G & Alnemri ES. In vitroactivation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains, Proc Natl Acad Sci USA, 1996, 93, 7464–7469.
26 Srinivasula SM, Fernandes-Alnemri T, Zangrilli J, Robertson N, Armstrong R, Wang L, Trapani JA, Tomaselli KJ, Litwack G & Alnemri ES. The Ced-3/interleukin 1β converting enzyme–like homolog Mch6 and the lamin-cleaving enzyme Mch2 are substrates for the apoptotic mediator CPP32, J Biol Chem, 1996, 271, 27099–27106.
27 Enari M, Talanian RV, Wong WW & Nagata S. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis, Nature, 1996, 380, 723–726.[Medline]
28 Thornberry NA, Peterson EP, Zhao JJ, Howard AD, Griffin PR & Chapman KT. Inactivation of interleukin-1β converting enzyme by peptide (acyloxy)methyl ketones, Biochemistry, 1994, 33, 3934–3940.[Medline]
29 Martins LM, Kottke T, Mesner PW, Basi GS, Sinha S, Frigon NJ, Tatar E, Tung JS, Bryant K, Takahashi A et al.. Activation of multiple interleukin-1β converting enzyme homologues in cytosol and nuclei of HL-60 human leukemia cells during etoposide-induced apoptosis, J Biol Chem, 1997, 272, 7421–7430.
30 Faleiro L, Kobayashi R, Fearnhead H & Lazebnik Y. Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells, EMBO (Eur Mol Biol Organ) J, 1997, 16, 2271–2281.[Medline]
31 Clayton LK, Ghendler Y, Mizoguchi E, Patch RJ, Ocain TD, Orth K, Bhan AK, Dixit VM & Reinherz EL. T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection, EMBO (Eur Mol Biol Organ) J, 1997, 16, 2282–2293.[Medline]
32 Zhou Q, Snipas S, Orth K, Muzio M, Dixit VM & Salvesen GS. Target protease specificity of the viral serpin CrmA – analysis of five caspases, J Biol Chem, 1997, 272, 7797–7800.
33 Datta R, Kojima H, Banach D, Bump NJ, Talanian RV, Alnemri ES, Weichselbaum RR, Wong WW & Kufe DW. Activation of a CrmA-insensitive, p35-sensitive pathway in ionizing radiation-induced apoptosis, J Biol Chem, 1997, 272, 1965–1969.
34 Kamada S, Funahashi Y & Tsujimoto Y. Caspase-4 and caspase-5, members of the ICE/CED-3 family of cysteine proteases, are CrmA-inhibitable proteases, Cell Death Diff, 1997, 4, 473–478.[Medline]
35 Chinnaiyan AM, Orth K, O'Rourke K, Duan H, Poirier GG & Dixit VM. Molecular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of the CED-3–like apoptotic proteases, J Biol Chem, 1996, 271, 4573–4576.
36 MacFarlane M, Cain K, Sun XM, Alnemri ES & Cohen GM. Processing/activation of at least four interleukin-1 beta converting enzyme–like proteases occurs during the execution phase of apoptosis in human monocytic tumor cells, J Cell Biol, 1997, 137, 469–479.
37 Lazebnik YA, Cole S, Cooke CA, Nelson WG & Earnshaw WC. Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis, J Cell Biol, 1993, 123, 7–22.
38 Yang CH, Lambie EJ & Snyder M. NuMA: an unusually long coiled-coil related protein in the mammalian nucleus, J Cell Biol, 1992, 116, 1303–1317.
39 Takahashi, A., E.S. Alnemri, T. Fernandes-Alnemri, Y.A. Lazebnik, R.D. Moir, R.D. Goldman, G.G. Poirier, S.H. Kaufmann, and W.C. Earnshaw. 1997. Biochemical dissection of nuclear events in apoptosis. In Cell Cycle Regulation. B. Metcalf, R.R.J. Ruffolo, and G. Poste, editors. Harwood Academic Publishers, Berks, UK. 131–149.
40 Kamada S, Washida M, Hasegawa J, Kusano H, Funahashi Y & Tsujimoto Y. Involvement of caspase-4 (–like) protease in Fas-mediated apoptotic pathway, Oncogene, 1997, 15, 285–290.[Medline]
41 Liu X, Kim CN, Pohl J & Wang X. Purification and characterization of an interleukin-1β–converting enzyme family protease that activates cysteine protease P32 (CPP32), J Biol Chem, 1996, 271, 13371–13376.
42 Ghayur T, Hugunin M, Talanian RV, Ratnofsky S, Quinlan C, Emoto Y, Pandey P, Datta R, Huang YY, Kharbanda S et al.. Proteolytic activation of protein kinase C delta by an ICE/CED 3–like protease induces characteristics of apoptosis, J Exp Med, 1996, 184, 2399–2404.
43 Han ZY, Hendrickson EA, Bremner TA & Wyche JH. A sequential two-step mechanism for the production of the mature p17:p12 form of caspase-3 in vitro, J Biol Chem, 1997, 272, 13432–13436.
44 Slee EA, Zhu H, Chow SC, MacFarlane M, Nicholson DW & Cohen GM. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32, Biochem J, 1996, 315, 21–24.[Medline]
45 Greidinger EL, Miller DK, Yamin TT, Casciola RL & Rosen A. Sequential activation of three distinct ICE-like activities in Fas-ligated Jurkat cells, FEBS Lett, 1996, 390, 299–303.[Medline]
46 Casiano CA, Martin SJ, Green DR & Tan EM. Selective cleavage of nuclear autoantigens during CD95 (Fas/ APO-1)–mediated T cell apoptosis, J Exp Med, 1996, 184, 765–770.
47 Gueth-Hallonet C, Weber K & Osborn M. Cleavage of the nuclear matrix protein NuMA during apoptosis, Exp Cell Res, 1997, 233, 21–24.[Medline]
48 Duan H, Chinnaiyan AM, Hudson PL, Wing JP, He W-W & Dixit VM. ICE-LAP3, a novel mammalian homologue of the Caenorhabditis eleganscell death protein Ced-3 is activated during Fas- and tumor necrosis factor– induced apoptosis, J Biol Chem, 1996, 271, 1621–1625.
49 Krajewska M, Wang HG, Krajewski S, Zapata JM, Shabaik A, Gascoyne R & Reed JC. Immunohistochemical analysis of in vivo patterns of expression of CPP32 (Caspase-3), a cell death protease, Cancer Res, 1997, 57, 1605–1613.
50 Nakagawara A, Nakamura Y, Ikeda H, Hiwasa T, Kuida K, Su MS-S, Zhao H, Cnaan A & Sakiyama S. High levels of expression and nuclear localization of interleukin-1β converting enzyme (ICE) and CPP32 in favorable human neuroblastomas, Cancer Res, 1997, 57, 4578–4584.
51 Lazebnik YA, Takahashi A, Moir R, Goldman R, Poirier GG, Kaufmann SH & Earnshaw WC. Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution, Proc Natl Acad Sci USA, 1995, 92, 9042–9046.
52 Takahashi A, Musy P-Y, Martins LM, Poirier GG, Turner PC, Moyer RW & Earnshaw WC. CrmA/ SPI-2 inhibition of an endogenous ICE-related protease responsible for lamin A cleavage and apoptotic nuclear fragmentation, J Biol Chem, 1996, 271, 32487–32490.
53 Takahashi A, Goldschmidt-Clermont PJ, Alnemri ES, Fernandes-Alnemri T, Yoshizawa-Kumagaya K, Nakajima K, Sasada M, Poirier GG & Earnshaw WC. Inhibition of ICE-related proteases (caspases) and nuclear apoptosis by phenylarsine oxide, Exp Cell Res, 1997, 231, 123–131.[Medline]
54 Rudel T & Bokoch GM. Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation of PAK2, Science, 1997, 276, 1571–1574.
55 Kroemer G, Petit P, Zamzami N, Vayssiere JL & Mignotte B. The biochemistry of programmed cell death, FASEB J, 1995, 9, 1277–1287.[Abstract]
56 Kroemer G, Zamzami N & Susin SA. Mitochondrial control of apoptosis, Immunol Today, 1997, 18, 44–51.[Medline]
57 Susin SA, Zamzami N, Castedo M, Daugas E, Wang HG, Geley S, Fassy F, Reed JC & Kroemer G. The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95– and ceramide–induced apoptosis, J Exp Med, 1997, 186, 25–37.
58 Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL & Henson PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages, J Immunol, 1992, 148, 2207–2216.[Abstract]
59 Emoto K, Toyama-Sorimachi N, Karasuyama H, Inoue K & Umeda M. Exposure of phosphatidylethanolamine on the surface of apoptotic cells, Exp Cell Res, 1997, 232, 430–434.[Medline]
60 Vayssiere JL, Petit PX, Risler Y & Mignotte B. Commitment to apoptosis is associated with changes in mitochondrial biogenesis and activity in cell lines conditionally immortalized with simian virus 40, Proc Natl Acad Sci USA, 1994, 91, 11752–11756.
61 Martin SJ, Reutelingsperger CP, McGahon AJ, Rader JA, van Schie RC, LaFace DM & Green DR. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl, J Exp Med, 1995, 182, 1545–1556.
62 Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng T-I, Jones DP & Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked, Science, 1997, 275, 1129–1132.
63 Kluck RM, Bossy-Wetzel E, Green DR & Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis, Science, 1997, 275, 1132–1136.
64 Chinnaiyan AM, O'Rourke K, Lane BR & Dixit VM. Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death, Science, 1997, 275, 1122–1126.
65 Kuida K, Zheng TS, Na S, Kuan C-Y, Yang D, Karasuyama H, Rakic P & Flavell RA. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice, Nature, 1996, 384, 368–372.[Medline]
66 Fernandes-Alnemri T, Takahashi A, Armstrong R, Krebs J, Fritz L, Tomaselli KJ, Wang L, Yu Z, Croce CM, Salveson G et al.. Mch3, a novel human apoptotic cysteine protease highly related to CPP32, Cancer Res, 1995, 55, 6045–6052.
67 Earnshaw WC. Nuclear changes in apoptosis, Curr Opin Cell Biol, 1995, 7, 337–343.[Medline]
68 Verhoven B, Schlegel RA & Williamson P. Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes, J Exp Med, 1995, 182, 1597–1601.
69 Zhou QS, Zhao J, Stout JG, Luhm RA, Wiedmer T & Sims PJ. Molecular cloning of human plasma membrane phospholipid scramblase— a protein mediating transbilayer movement of plasma membrane phospholipids, J Biol Chem, 1997, 272, 18240–18244.
70 Casciola-Rosen LA, Anhalt G & Rosen A. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes, J Exp Med, 1994, 179, 1317–1330.
71 Toyoshima F, Moriguchi T & Nishida E. Fas induces cytoplasmic apoptotic responses and activation of the MKK7-JNK/SAPK and MKK6-p38 pathways independent of CPP32-like protease, J Cell Biol, 1997, 139, 1005–1015.
72 Talanian RV, Quinlan C, Trautz S, Hackett MC, Mankovich JA, Banach D, Ghayur T, Brady KD & Wong WW. Substrate specificities of caspase family proteases, J Biol Chem, 1997, 272, 9677–9682.
73 Thornberry NA, Rano TA, Peterson EP, Rasper DM, Timkey T, Garcia-Calvo M, Houtzager VM, Nordstrom PA, Roy S, Vaillancourt JP et al.. A combinatorial approach defines specificities of members of the caspase family and granzyme B – functional, relationships established for key mediators of apoptosis, J Biol Chem, 1997, 272, 17907–17911.
74 Pai J-T, Brown MS & Goldstein JL. Purification and cDNA cloning of a second apoptosis-related cysteine protease that cleaves and activates sterol regulatory element binding proteins, Proc Natl Acad Sci USA, 1996, 93, 5437–5442.
75 Antosson B, Conti F, Ciavatta A, Montessuit S, Lewis S, Martinou I, Bernasconi L, Bernard A, Mermod J-J, Mazzei G et al.. Inhibition of Bax channel-forming activity by Bcl-2, Science, 1997, 277, 370–372.
76 Susin SA, Zamzami N, Castedo M, Hirsch T, Marchetti P, Macho A, Daugas E, Geuskens M & Kroemer G. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease, J Exp Med, 1996, 184, 1331–1341.
77 Takahashi, A., and W.C. Earnshaw. 1997. In vitro systems for the study of apoptosis. In Apoptosis: Pharmacological Implications and Therapeutic Opportunities. S.H. Kaufmann, editor. Academic Press, San Diego. 89–106.
78 Thompson CB. Apoptosis in the pathogenesis and treatment of disease, Science, 1995, 267, 1456–1462.
79 Fisher DE. Apoptosis in cancer therapy: crossing the threshold, Cell, 1994, 78, 539–542.[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| TABLE OF CONTENTS |
|
