The Role of Calcium in the Regulation of Apoptosis

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

239, 357–366 (1997)

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BREAKTHROUGHS AND VIEWS The Role of Calcium in the Regulation of Apoptosis David J. McConkey*,1 and Sten Orrenius† *Department of Cell Biology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas; and †Division of Toxicology, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden

Received August 22, 1997

Apoptosis (programmed cell death) has gained widespread attention due to its roles in a variety of physiological and pathological processes, yet precisely how apoptosis is regulated by external and internal cues remains unclear. Work from our laboratories and others has implicated alterations in intracellular Ca2/ in apoptosis, and more recent work has defined particular biochemical processes that are targeted by Ca2/ in apoptotic cells. This review will summarize the role of Ca2/ in apoptosis within the context of what is known about the core components of the effector machinery for apoptosis. q 1997 Academic Press

Although the term ‘‘apoptosis’’ was originally coined to describe a series of morphological alterations associated with particular examples of programmed cell death (1), it is now generally used to describe the evolutionarily conserved pathway of biochemical and molecular events that presumably underlies these changes. Work in the nematode Caenorhabditis elegans made identifying the molecular components of this pathway possible, where mutagenesis studies demonstrated that 3 genes, designated ced-3, ced-4, and ced-9, were centrally involved in controlling programmed cell death. Ced-3 is a member of a growing family of cysteine proteases that are homologous to human interleukin 1b converting enzyme (ICE), now collectively termed caspases, that function as components of the effector machinery for cell death. Caspase activation is promoted by release of cytochrome c from mitochondria (2), suggesting that activation of the apoptotic pathway is coupled to loss of mitochondrial function. Ced-9 encodes an inhibitor of apoptosis in the nematode that is a member of the bcl-2 gene family, which in mamma1 Address correspondence to David McConkey, Box 173, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. Fax: (713) 792-8747. E-mail: [email protected].

lian cells includes both inhibitors and inducers of apoptosis (3). These proteins act upstream of the caspases in the pathway, controlling their activation. Finally, very recent work has identified a mammalian homolog for ced-4, which apparently promotes cytochrome c-dependent activation of the caspases (4) and can physically interact with ced-9 and other members of the bcl-2 gene family (5, 6). Precisely how the 3 gene products regulate one another during apoptosis is under intensive investigation, but the binding studies alluded to above suggest that CED-4 might represent a convergence point for signals for cell death. Endonuclease activation is another important biochemical event in apoptosis, although the polypeptides involved remain unclear. Early work demonstrated that the chromatin in apoptotic cells is fragmented nonrandomly into integer multiples of 180-200 base pairs suggestive of cleavage between nucleosomes (7), and these ‘‘DNA ladders’’ remain a standard means of demonstrating apoptotic cell death. However, more recent work has shown that the formation of larger DNA fragments, most notably of 300 kilobases and 50 kilobases in size (8), precedes oligonucleosomal DNA fragmentation in all systems investigated (9), and in some cases cells may die by apoptosis without forming DNA ladders at all (10). The biochemical mechanisms underlying this stepwise chromatin cleavage remain unclear, but it appears that chromatin topology rather than sequence specificity of the endonuclease is involved (8). It is possible that the discrete fragment sizes are generated as particular chromatin-associated proteins (topoisomerase II, lamin B, and histone H1) are sequentially cleaved by the caspases and perhaps other protease(s) during apoptosis (11, 12), thereby exposing these regions of chromatin to endonuclease action. A link between the caspases and the endonuclease is demonstrated by the observation that caspase inhibitors block DNA fragmentation and that endonuclease activation in isolated nuclei can be promoted by a heterodimeric caspase substrate (DFF) (13).

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Less is known about events leading to disruption of mitochondrial function following exposure to the diverse stimuli that trigger apoptosis, with cell death induced by engagement of the CD95/Fas/APO-1 molecule representing the one notable exception. Fas was originally identified by two independent laboratories as a cell surface molecule capable of triggering rapid cell death upon multivalent crosslinking with monoclonal antibodies (14). Subsequent cloning of the target antigen revealed it to be homologous to the tumor necrosis factor receptor (TNF-R) superfamily, and its ligand (FasL) is a member of the TNF superfamily (14). The structural requirements for Fas-mediated apoptosis have been defined, and a region within its carboxyterminus known as the ‘‘death domain’’ (DD) is critical for function. This motif binds an adaptor protein (FADD) (15) which then recruits one of the caspases (caspase-8, or FLICE) to the death-inducing signaling complex (DISC) that is assembled at the cytosolic surface of the plasma membrane (16). The interaction between FADD and FLICE is mediated by another polypeptide motif, termed the death effector domain (DED), that is found in both proteins. Because FLICE (and the other caspases) can be activated by autocatalytic proteolytic processing, it is likely that the formation of the DISC is sufficient to trigger caspase activation. Thus, Fas is directly coupled to the effector machinery for apoptosis. Interestingly, other cell surface molecules (most notably the type I TNFR) also possess cytoplasmic DD’s, and their abilities to trigger apoptosis could be due to the formation of similar DISCs. Although activation of the Fas pathway may be widely involved in triggering apoptosis in response to physiological stress, there are presumably other means of activating the caspases and endonucleases and triggering apoptosis in mammalian cells. We and others have shown that alterations in intracellular Ca2/ homeostasis are commonly observed during apoptosis, and insights into how Ca2/ might regulate the caspases and other components of the pathway are emerging. This review will summarize what is known about the involvement of Ca2/ in apoptosis, focusing on how the Ca2/ alterations occur and their potential biochemical consequences. CALCIUM ELEVATIONS IN APOPTOSIS Early studies by Kaiser and Edelman demonstrated that glucocorticoid-stimulated thymocyte apoptosis is associated with enhanced Ca2/ influx (17), observations we have since confirmed using other techniques (18). However, intracellular Ca2/ storage sites also appear to be affected, as the Ca2/ pool located in the endoplasmic reticulum is depleted in a lymphoid cell line in response to glucocorticoid treatment (19), and a similar phenomenon has been documented in an interleukin 3 (IL-3)dependent myeloid cell line undergoing apoptosis fol-

lowing IL-3 withdrawal (20). Indirect evidence suggests that the mitochondrial Ca2/ pool may also be affected (21), as a drop in mitochondrial membrane potential precedes the Ca2/ increase in several models of apoptosis (22, 23). Rapid, sustained Ca2/ increases precede the cytolysis of the targets of cytotoxic T lymphocytes (24) and natural killer (NK) cells (25). In developing T lymphocytes high affinity engagement of the T cell receptor induces apoptosis (26) that involves a sustained Ca2/ elevation (27, 28). Both second messenger- and damage-mediated mechanisms can be involved in promoting Ca2/ increases in apoptotic cells. In an example of the former, T cell receptor engagement on thymocytes leads to a sustained increase in the cytosolic Ca2/ concentration that involves protein tyrosine kinase activation, phosphorylation of the g-isoform of phospholipase C, phosphoinositide hydrolysis leading to the production of inositol trisphosphate (IP3), and mobilization of Ca2/ from the endoplasmic reticulum and extracellular millieu that promote cell death. Similarly, surface antigen receptor engagement on B cells leads to Ca2/ increases that promote cell death (29, 30). Thus, in these examples of apoptosis the initial Ca2/ increases occur via a controlled, physiological mechanism that is also utilized in alternative responses such as cellular activation leading to proliferation. Disruption of mitochondrial function and subsequent oxidative stress can also induce an increase in Ca2/. The cytosolic Ca2/ concentration is maintained at roughly 100 nM in resting cells, whereas the concentrations in the extracellular millieu and the ER are estimated to be much higher (in the millimolar range). Early work on the biochemical mechanisms underlying the cytotoxicity of agents that generate reactive oxygen species in cells (oxidative stress) indicated that the Ca2/ transport systems located within the ER, mitochondria, and plasma membrane can be damaged by oxygen radicals that are largely derived from mitochondria as the result of uncoupling of oxidative phosphorylation (21, 31). This leads to diffusion of Ca2/ down its concentration gradient, a disruption of intracellular Ca2/ homeostasis, and sustained Ca2/ increases. We and others have shown that antioxidants and inhibitors of the mitochondrial transition pore block the glucocorticoid-induced Ca2/ increase observed in thymocytes (22, 32), suggesting that this type of mechanism also participates in apoptosis. Direct evidence that Ca2/ increases are important for apoptosis has been obtained from experiments with intracellular Ca2/ buffering agents and extracellular Ca2/ chelators, which can inhibit caspase activation (J. Chandra and D.J. McConkey, manuscript submitted), DNA fragmentation, and cell death (33). The Ca2/-dependent regulatory cofactor calmodulin may link these Ca2/ alterations to the effector machinery, as calmodulin antagonists can interfere with apoptosis in some

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of these systems (18, 34) and increases in calmodulin expression are linked to apoptosis in glucocorticoidtreated thymoma cells (34) and in prostatic epithelial cells following withdrawal of androgen (35). Independent evidence for the involvement of Ca2/ influx in the triggering of apoptosis has come from studies with specific Ca2/ channel blockers, which abrogate apoptosis in the regressing prostate following testosterone withdrawal (36) and in pancreatic b-cells treated with serum from patients with type I diabetes (37). Other support for the involvement of Ca2/ in apoptosis comes from the observation that agents which directly mobilize Ca2/ can trigger apoptosis in diverse cell types. Early work by Kaiser and Edelman demonstrated the cytolytic effects of glucocorticoids on lymphoid cells can be mimicked by treating the cells with Ca2/ ionophores (38). Subsequently, Wyllie and coworkers demonstrated that Ca2/ ionophores cause endonuclease activation as well as many of the morphological changes that are typical of apoptosis in thymocytes (39). Calcium ionophores also trigger apoptosis in prostate tumor cells (36). Other support for this mechanism has come from studies with the endoplasmic reticular Ca2/-ATPase inhibitor thapsigargin, the product of the plant, Thapsa garganica, which can also trigger all of the morphological and biochemical events of apoptosis in thymocytes (40) and some other cell types (41-43). CALCIUM COUPLING TO THE EFFECTOR PATHWAY An important aspect of ongoing research involves defining the biochemical consequences of Ca2/ mobilization in apoptotic cells, and at present there are two models to explain how these alterations might trigger apoptosis. In one, depletion of intracellular stores and possibly influx of Ca2/ across the plasma membrane promote a sustained Ca2/ increase that acts as a signal for apoptosis, perhaps in part by activating key catabolic enzymes that make up parts of the effector machinery. In the second, it is not the Ca2/ increase but the emptying of intracellular Ca2/ stores that triggers apoptosis, perhaps by disrupting intracellular architecture and allowing key elements of the effector machinery to gain access to their substrates. These models are certainly not mutually exclusive. Evidence for both models will be presented below, but it should be emphasized at the outset that definitive proof for either one is lacking at present. POSSIBLE TARGETS FOR Ca2/ ELEVATIONS 1. Signal Transduction Intermediates Activation of Ca2/ -dependent protein kinases and/ or phosphatases leading to alterations in gene tran-

scription is one way Ca2/ might regulate apoptosis. Most support for this hypothesis has come from experiments with the immunosuppressant cyclosporin A, a compound that binds a family of cytosolic receptors termed cyclophilins and in so doing forms a composite molecular surface that binds to and inhibits the Ca2// calmodulin-dependent protein serine/threonine phosphatase, calcineurin (44). Cyclosporin A can block Ca2/-dependent apoptosis in lymphoid model systems (45-47), indicating that calcineurin activation may be required for these responses. Induction of the orphan steroid receptor Nur77 and the Fas ligand are likely molecular targets of cyclosporin A in mature T cells and T cell hybridomas (48, 49). Because calcineurin is known to activate the transcription factor, NF-AT (nuclear factor of activated T cells), it is possible that the effects of cyclosporin A involve suppression of NFAT function. Supporting this idea, recent work suggests that NF-AT is activated during Ca2/ dependent apoptosis, and BCL-2 suppression of these responses is linked to direct binding to calcineurin and inhibition of its function (50). 2. Ca2/-Activated Proteases Direct activation of the Ca2/ dependent neutral protease, calpain, appears to represent another target for Ca2/ action in apoptosis. Calpain is rapidly activated in apoptotic thymocytes (51) and neuronal cells (52), and specific inhibitors of calpain block various features of apoptosis in a number of different systems (51, 53-55). The cytoskeletal protein, fodrin, appears to be one of the substrates for calpain in apoptotic cells but the polypeptide can also be cleaved by the caspases (54). Furthermore, calpain-mediated cleavage of fodrin can also be observed during necrosis (54), suggesting that the event is not selective for one mode of cell death. The nuclear scaffold (NS) protease is another Ca2/ sensitive enzyme that is activated in apoptosis. Initial work implicated the protease in the rapid, Ca2/ stimulated degradation of the lamins in isolated nuclei (56, 57). Lamin cleavage in apoptotic thymocytes and isolated thymocyte nuclei also occurs via a Ca2/ dependent mechanism (12). Supporting this notion, peptide inhibitors of the NS protease block lamin cleavage in thymocytes (58-60) and in isolated B lymphocytes from patients with chronic lymphocytic leukemia (CLL) (61). Moreover, these inhibitors also block cellular shrinkage and DNA fragmentation in thymocytes and CLL cells, indicating that the NS protease is also required for endonuclease activation. Importantly, however, the lamins can also be cleaved by caspases (13, 62), suggesting that two separate pathways to lamin cleavage may exist in apoptotic cells. In more recent work we have investigated whether the NS protease interacts functionally with caspases.

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Establishing where the NS protease acts within the apoptotic pathway has been more problematic. In thymocytes, inhibitors of the NS protease prevent caspase activation, consistent with a role for the protease upstream of the caspases (J. Chandra, D.J. McConkey, manuscript submitted). However, in CLL lymphocytes, NS protease inhibitors actually promote caspase activation, even though they still completely prevent DNA fragmentation (61). Interestingly, in both systems NS protease inhibitors (and intracellular Ca2/ chelators) directly induce loss of mitochondrial membrane potential and exposure of phosphatidylserine (another early marker of apoptosis, discussed below). In ongoing work we are purifying the protease in order to define its role in apoptosis more precisely, but preliminary evidence suggests that the protease is functionally linked to the proteasome, a large, multisubunit complex that mediates a diverse array of physiological functions. Another group reported the isolation of a candidate 29 kD component of the protease from rat liver nuclei, and microsequencing revealed it to be highly homologous to a human proteasome b subunit (56). Our own preliminary work has identified a single, 130 kD polypeptide in thymocytes that interacts with inhibitors of the NS protease; the size of this protein is inconsistent with the core components of the 20S proteasome, which are all between 20-35 kD in molecular weight. However, using antibodies specific to a surface epitope on the proteasome b chain, we have obtained evidence that our polypeptide can be co-immunoprecipitated with the proteasome in thymocytes (M. Harbison, J. Chandra, D.J. McConkey, unpublished observations). A role for the proteasome in apoptosis is consistent with previous work implicating the ubiquitin/proteasome pathway in programmed cell death in the moth, Manduca sexta (63), and with more recent work demonstrating that proteasome inhibitors block caspase activation and DNA fragmentation in apoptotic thymocytes (64) and neuronal cells (65). 2/

3. Ca -Activated Endonuclease(s) As introduced above, endonuclease activation resulting in the formation of oligonucleosome-length DNA fragments (DNA ladders) remains the most characteristic biochemical feature of apoptotic cell death. Early work by Hewish and Burgoyne (66) and later by Vanderbilt and colleagues (67) demonstrated that a Ca2//Mg2/-dependent enzyme activity capable of generating characteristic apoptotic chromatin cleavage patterns is constitutively present within nuclei of a variety of different cell types. Subsequent work by Cohen and Duke (68), and Wyllie and coworkers(39), demonstrated the involvement of this activity in the DNA fragmentation observed in thymocytes undergoing apoptosis, and it is now thought that it mediates DNA fragmentation in a variety of other model systems as

well. The search for and purification of potential Ca2/ dependent apoptotic nucleases has subsequently been undertaken by several laboratories. Thus, Gaido and Cidlowski (69) have described a low-molecular weight nuclease (NUC18) with Ca2/ and Mg2/ dependence activity in apoptotic lymphoid cells in response to several kinds of apoptotic stimuli. (Interestingly, the purified NUC18 shares amino acid sequence homology with cyclophilin, and human recombinant cyclophilin A has biochemical and pharmacological properties identical to native NUC18 (70).) NUC18 is also present in untreated thymocytes in precursor form or as part of a higher molecular weight complex (ú100 kDa. Although the precise mechanism of liberation of active enzyme from its precusors is unknown, an attractive possibility is that it may involve proteolysis. The Ca2/-dependent endonuclease DNase I is another excellent candidate apoptotic nuclease (71). Addition of the enzyme to isolated nuclei and other reconstitution systems promotes the formation of DNA strand breaks that possess the same 5*-PO4 and 3*-OH end groups found in DNA fragments isolated from apoptotic cells. Although the enzyme is localized within the rough endoplasmic reticulum, the Golgi complex, and small (secretory) vesicles in viable cells, it is also found within the perinuclear space of apoptotic cells, and it is possible that structural alterations in the ER and/or nuclear envelope associated with apoptosis may promote the entry of DNase I into the nucleus (see below). A similar mechanism may promote entry of an ERlocalized fraction of the NS lamin protease into the nucleus. Several other proteins with Ca2//Mg2/ endonuclease activity have been isolated (72-75), but to date proof that any one of these activities is directly involved in oligonucleosomal DNA fragmentation in apoptosis is lacking. 4. Transglutaminase Activation Transglutaminases are a group of Ca2/-dependent enzymes that catalyze the post-translational coupling of amines (including polyamines) to proteins and the crosslinking of proteins via gamma glutamyl lysine bridges when the amine is a peptide-bound lysine residue. Tissue transglutaminase has been implicated in a number of physiological processes, including crosslinking of integral plasma membrane proteins with the cytoskeleton. Recent work indicates that tissue transglutaminase is also involved in induction of apoptosis (76, 77). Expression of transglutaminase mRNA and protein levels increase markedly in dying cells. The enzyme appears to be activated by elevations of the cytosolic Ca2/ concentration. Isolation of apoptotic bodies from a number of different tissues has shown that they are resistant to dissolution by detergents and chaotrophic agents; this may in part be explained by the fact that surface polypeptides in these structures

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are crosslinked via gamma glutamyl lysine isopeptide bonds (78). The resistance of these structures to proteolysis may allow them to accumulate, and they can be detected in the media of cell cultures containing high rates of apoptotic cell death (79). Isodipeptide can also be detected in normal plasma, and its concentration increases following induction of apoptosis in various organs, including the thymus and liver. Efforts are underway to determine whether serum isodipeptide levels can be used to estimate responses to chemotherapy in cancer patients. The role of transglutaminase promoting cell death and/or phagocytosis is still poorly understood. One possibility is that protein crosslinking stabilizes apoptotic cells and bodies, preventing leakage of intracellular contents into the extracellular millieu (which can trigger inflammation). Alternatively, transglutaminase modification may target proteins for subsequent degradation. Intriguingly, overexpression of the enzyme has been reported to trigger apoptotic cell death (80), suggesting that transglutaminase may be a component of the death effector pathway. Further efforts are required to identify the substrates for transglutaminase in apoptotic cells and to determine the consequences of their modification. 5. Exposure of Phosphatidylserine and Macrophage Recognition Recent work indicates that the movement of phosphatidylserine (PS) from the inner to the outer surface of the plasma membrane, a process that functions in the removal of apoptotic cells and bodies by both professional phagocytic cells (macrophages) and neighboring cells in tissues (81, 82), is another component of apoptosis that appears to be regulated by alterations in cytosolic Ca2/. Phospholipids are known to be distributed asymmetrically across the plasma membrane, with phosphatidylcholine and sphingomyelin localized primarily to the extracellular surface and PS and phosphatidylethanolamine restricted almost exclusively to the intracellular surface under normal conditions (83, 84). Most of the work on plasma mebrane lipid asymmetry has been conducted with red blood cells, where it is known that PS localization is regulated by energy-dependent processes involving specific lipid transporters that move PS to the outside or inside surface, respectively. Transport in both directions is ATP-dependent and sensitive to sulfhydryl modifying agents. Interestingly, inhibition of lipid movement with reagents that abrogate energy-dependent transport does not result in loss of membrane asymmetry (85), suggesting that these enzymes may primarily function to restore lipid asymmetry following its disruption. However, transport can also be inhibited by increasing the cytosolic Ca2/ concentration, which does result in rapid non-specific redistribution of all phospholipids (86). The mechanism of Ca2/ -

mediated PS exposure is not yet clear, but in red blood cells it closely parallels formation of cytoskeleton-free lipid microvesicles (87) and other phenomena such as calpain activation (88) and the formation of phosphatidylinositol(4,5)-bisphosphate-Ca2/ complexes (89). However, neither direct Ca2/ effects, calpain-mediated protein degradation (53, 90), Ins(4,5)P2 accumulation (91), nor inhibition of the flipase can singularly accomodate the membrane rearrangements that occur. More recent work suggests that Ca2/ -mediated redistribution is mediated by a Ca2/ -and sulfhydryl-sensitive, energy-dependent lipid scramblase (92). Therefore, elevations in the cytosolic Ca2/ concentration probably promote PS exposure and allow for macrophage recognition primarily by inactivating the PS translocase and by activating the scramblase (93). The source of this Ca2/ appears to be extracellular, as demonstrated by the observation that intracellular Ca2/ chelators fail to block PS exposure ((53), J. Chandra and D.J. McConkey, manuscript submitted). Interestingly, PS exposure on aged red blood cells is associated with increased cytosolic Ca2/ levels and increased cell density, suggesting that these events may be mechanistically related in both red cells and apoptotic cells. In addition, given its regulation by Ca2/and calpain-dependent mechanisms, it is tempting to speculate that red blood cell microvesiculation may be functionally related to the formation of apoptotic bodies by nucleated cells undergoing apoptosis. Plasma membrane lipid asymmetry is critically involved in several other important physiological functions. For example, surface-exposed PS serves as the point of assembly for the coagulation factors Va and Xa into the prothrombinase complex (94). In addition, PS exposure enhances membrane fusion events and appears to be involved in the initiation of microvesiculation in red blood cells(95). Finally, surface PS is also detectable on certain tumor cells (96). Although the mechanisms underlying the latter have not been defined, it is possible that PS exposure is involved in the prominent macrophate infiltration observed in most solid tumors and that surface PS may represent a potential target for anti-tumor therapies. POSSIBLE CONSEQUENCES OF INTRACELLULAR Ca2/ POOL DEPLETION In some cellular systems, extracellular or intracellular Ca2/ chelators can actually promote DNA fragmentation, even though other triggers of apoptosis in these systems (i.e. glucocorticoids, growth factor withdrawal) have been shown to deplete the ER Ca2/ store. These observations have led Baffy and colleagues (20) and more recently Lam and coworkers (19) to propose that depletion of the ER Ca2/ store may itself serve as a signal for apoptosis. How could this occur? At least two of the catabolic enzymes proposed to be involved in the effector mechanism of apoptosis (DNase I and an

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extranuclear pool of the NS protease) are localized to the ER, and it is therefore possible that loss of Ca2/ leads to release of these factors into the perinuclear region or into the nuclear matrix itself. In addition, it is known that ER Ca2/ pool depletion results in the release of a small biomolecule that partipates in a retrograde signal for plasma membrane Ca2/ influx, and it is possible that it or another molecule released in a similar fashion can also promote cell death. Depletion of mitochondrial Ca2/ stores may also partipate in the signal for apoptosis. Mitochondrial Ca2/ uptake is driven by mitochondrial membrane potential (DC)(21). In de-energized mitochondria Ca2/ can be released by a reversal of the uptake pathway. Under conditions of oxidative stress, mitochondrial Ca2/ cycling can reach critical levels, leading to increased energy expenditure and a dramatic fall in DC. Recent work has shown that a fall in mitochondrial DC is an early event in apoptosis (97-99), and ruthenium red, an inhibitor of the mitochondrial Ca2/ uptake pathway, blocks apoptosis in L929 fibroblasts (100) and inhibits the progression of apoptosis in glucocorticoid-treated splenocytes, suggesting that mitochondrial Ca2/ release is involved. Again, further efforts are required to determine the relationship between this event and the activation of the proteases and nucleases of the effector pathway. CALCIUM REGULATION BY BCL-2 Investigation into the biochemical mechanisms underlying BCL-2 family functions has demonstrated that BCL-2 regulates intracellular Ca2/ compartmentalization. Work by Baffy and colleagues showed that BCL-2 can block the depletion of the endoplasmic reticular Ca2/ pool in transfectants of an interleukin 3-dependent cell line (32D) (20). Interestingly, these authors also demonstrated that constitutive levels of Ca2/ in mitochondria (measured following treatment with an uncoupler that promotes rapid and selective depletion of this intracellular Ca2/ store) were significantly lower in BCL-2-expressing cells compared to vector control transfectants, consistent with the notion that BCL-2 may also regulate Ca2/ compartmentalization in mitochondria. More recently, Lam and coworkers have shown that overexpression of BCL-2 interferes with thapsigargin-induced Ca2/ mobilization from the ER in the WEHI7.2 T lymphoma cell line, an effect that is associated with preservation cell viability (101). Finally, we have shown that BCL-2 blocks accumulation of Ca2/ in the nucleus in cells exposed to either tumor necrosis factor or thapsigargin (102). Precisely how BCL-2 regulates intracellular Ca2/ is still unclear, although recent work demonstrating that BCL-2 and its homologs can form transmembrane ion channels could be relevant. Crystallographic analysis of the core domains of BCL-XL revealed it to be

homologous to certain bacterial pore-forming toxins (103), and studies with planar membrane bilayers confirmed that recombinant BCL-X L , BCL-2, and BAX possess pH-sensitive pore-forming activities (3). These channels may be somewhat cation-selective at neutral pH, although it is thought that they may be large enough to accomodate larger molecules, including small proteins. Because no structure-function analyses have been conducted, the polypeptide domain(s) involved in these activities are not known, nor is it clear that pore formation is required for inhibition of apoptosis by BCL-2 and BCL-XL or stimulation of apoptosis by BAX. However, recent work demonstrating that BAX-induced channels are antagonized by BCL-2 may account for the in vivo differences in function observed (104). It is possible that BAX and the other pro-apoptotic members of the family facilitate Ca2/ efflux from the ER and Ca2/ influx into the nucleus, effects that are prevented by BCL-2. BAX may also promote release of cytochrome c from mitochondria, as it has already been established that BCL-2 can block this event (105, 106). CONCLUSIONS AND FUTURE DIRECTIONS From the results summarized above it is clear that Ca2/ regulates several key steps in the apoptotic pathway, from early signaling events to the chromatin cleavage that appears to mark irreversible commitment to cell death. However, a clear picture of how Ca2/ exerts these effects at the molecular level is still not available. Important to future investigation will be to conclusively identify the Ca2/ -dependent endonuclease(s) responsible for cleavage of host chromatin and the Ca2/ dependent protease(s) that appear to regulate this event. In addition, precisely how these intermediates interact with the known core components of the apoptotic pathway (the caspases, the bcl-2 family, and ced-4) must be determined. Hopefully solutions to these problems will lead to the development of better strategies for modulating apoptotic cell death in the treatment of human pathologies such as cancer and neurodegenerative disorders. REFERENCES 1. Kerr, J. F. R., Wyllie, A. H., and Currie, A. R. (1972) Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer. 26, 239–257. 2. Liu, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. (1996) Induction of the apoptotic program in cell-free extracts: Requirement for dATP and cytochrome c. Cell 86, 147–157. 3. Reed, J. C. (1997) Double identity for proteins of the BCL-2 family. Nature 387, 773–776. 4. Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Apaf-1, a human protein homologous to C. elegans ced-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405–413.

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5. Chinnaiyan, A. M., O’Rourke, K., Lane, B. R., and Dixit, V. M. (1997) Interaction of ced-4 with ced-3 and ced-9: A molecular framework for cell death. Science 275, 1122–1126. 6. Wu, D., Wallen, H. D., and Nunez, G. (1997) Interaction and regulation of subcellular localization of ced-4 by ced-9. Science 275, 1126–1129. 7. Wyllie, A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556. 8. Filipski, J., Leblanc, J., Youdale, T., Sikorska, M., and Walker, P. R. (1990) Periodicity of DNA folding in higher order chromatin structures. EMBO J. 9, 1319–1327. 9. Brown, D. G., Sun, X. M., and Cohen, G. M. (1993) Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation. J. Biol. Chem. 268, 3037–3039. 10. Oberhammer, F., Wilson, J. W., Dive, C., Morris, I. D., Hickman, J. A., Wakeling, A. E., Walker, P. R., and Sikorska, M. (1993) Apoptotic death in epithelial cells: Cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12, 3679–3684. 11. Kaufmann, S. H. (1989) Induction of endonucleolytic DNA cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: A cautionary note. Cancer Res. 49, 5870–5878. 12. Neamati, N., Fernandez, A., Wright, S., Kiefer, J., and McConkey, D. J. (1995) Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei. J. Immunol. 154, 3788–3795. 13. Liu, X., Zou, H., Slaughter, C., and Wang, X. (1997) DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89, 175– 184. 14. Nagata, S. and Golstein, P. (1995) The Fas death factor. Science 267, 1449–1455. 15. Chinnaiyan, A. M., O’Rourke, K., Tewari, M., and Dixit, V. M. (1995) FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell 81, 505–512. 16. Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) FLICE, a novel FADD-homologous ICE/CED-3l,ike protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85, 817–827. 17. Kaiser, N. and Edelman, I. S. (1977) Calcium dependence of glucocorticoid-induced lymphocytolysis. Proc. Natl. Acad. Sci. USA. 74, 638–642. 18. McConkey, D. J., Nicotera, P., Hartzell, P., Bellomo, G., Wyllie, A. H., and Orrenius, S. (1989) Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic Ca2/ concentration. Arch. Biochem. Biophys. 269, 365–370. 19. Lam, M., Dubyak, G., and Distelhorst, C. W. (1993) Effect of glucocorticoid treatment on intracellular calcium homeostasis in mouse lymphoma cells. Mol. Endocrinol. 7, 686–693. 20. Baffy, G., Miyashita, T., Williamson, J. R., and Reed, J. C. (1993) Apoptosis induced by withdrawal of interleukin-3 (IL-3) from an IL-3-dependent hematopoietic cell line is associated with repartitioning of intracellular calcium and is blocked by enforced BCL-2 oncoprotein production. J. Biol. Chem. 268, 6511–6519. 21. Richter, C. (1993) Pro-oxidants and mitochondrial Ca2/: Their relationship to apoptosis and oncogenesis. FEBS Lett. 325, 104–107. 22. Macho, A., Hirsch, T., Marzo, I., Marchetti, P., Dallaporta, B.,

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33. 34.

35.

36.

37.

38.

39.

Susin, S. A., Zamzami, N., and Kroemer, G. (1997) Glutathione depletion is an early and calcium elevation is a late event of thymocyte apoptosis. J. Immunol. 158, 4612–4619. Backway, K. L., McCulloch, E. A., Chow, S., and Hedley, D. W. (1997) Relationships between the mitochondrial permeability transition and oxidative stress during ara-C toxicity. Cancer Res. 57, 2446–2451. Allbritton, N. L., Verret, C. R., Wolley, R. C., and Eisen, H. N. (1988) Calcium ion concentrations and DNA fragmentation in target cell destruction by murine cloned cytotoxic T lymphocytes. J. Exp. Med. 167, 514–527. McConkey, D. J., Chow, S. C., Orrenius, S., and Jondal, M. (1990) NK cell-induced cytotoxicity is dependent on a Ca2/ increase in the target. FASEB J. 4, 2661–2664. Smith, C. A., Williams, G. T., Kingston, R., Jenkinson, E. J., and Owen, J. J. T. (1989) Antibodies to CD3/T cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 337, 181–184. McConkey, D. J., Hartzell, P., Amador-Perez, J. F., Orrenius, S., and Jondal, M. (1989) Calcium-dependent killing of immature thymocytes by stimulation via the CD3/T cell receptor complex. J. Immunol. 143, 1801–1806. Nakagama, T., Ueda, Y., Yamada, H., Shores, E. W., Singer, A., and June, C. H. (1992) In vivo calcium elevations in thymocytes with T cell receptors that are specific for self ligands. Science 257, 96–99. Tsubata, T., Wu, J., and Honjo, T. (1993) B-cell apoptosis induced by antigen receptor crosslinking is blocked by a T-cell signal through CD40. Nature 364, 645–648. Yao, X. R. and Scott, D. W. (1993) Antisense oligodeoxynucleotides to the blk tyrosine kinase prevent anti-m-mediated growth inhibition and apoptosis in a B-cell lymphoma. Proc. Natl. Acad. Sci. USA. 90, 7946–7950. Orrenius, S., McConkey, D. J., Bellomo, G., and Nicotera, P. (1989) Role of Ca2/ in toxic cell killing. Trends Pharmacol. Sci. 10, 281–285. Fernandez, A., Kiefer, J., Fosdick, L., and McConkey, D. J. (1995) Oxygen radical production and thiol depletion are required for Ca2/-mediated endogenous endonuclease activation in apoptotic thymocytes. J. Immunol. 155, 5133–5139. McConkey, D. J. and Orrenius, S. (1996) Signal transduction pathways in apoptosis. Stem Cells 14, 619–631. Dowd, D. R., MacDonald, P. N., Komm, B. S., Maussler, M. R., and Miesfeld, R. (1991) Evidence for early induction of calmodulin gene expression in lymphocytes undergoing glucocorticoidmediated apoptosis. J. Biol. Chem. 266, 18423–18426. Furuya, Y., and Isaacs, J. T. (1993) Differential gene regulation during programmed death (apoptosis) versus proliferation of prostatic glandular cells induced by androgen manipulation. Endocrinology 133, 2660–2666. Martikainen, P., and Isaacs, J. (1990) Role of calcium in the programmed cell death of rat ventral prostatic glandular cells. Prostate 17, 175–187. Juntti-Berggren, L., Larsson, O., Rorsman, P., Ammala, C., Bokvist, K., Wahlander, K., Nicotera, P., Dybukt, J. M., Orrenius, S., Hallberg, A., and Berggren, P. (1993) Increased activity of L-type Ca2/ channels exposed to serum from patients with type I diabetes. Science 261, 86–90. Kaiser, N., and Edelman, I. S. (1978) Further studies on the role of calcium in glucocorticoid-induced lymphocytolysis. Endocrinology 103, 936–942. Wyllie, A. H., Morris, R. G., Smith, A. L., and Dunlop, D. (1984) Chromatin cleavage in apoptosis: Association with condensed chromatin morphology and dependence on macromolecular synthesis. J. Pathol. 142, 67–77.

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40. Jiang, S., Chow, S. C., Nicotera, P., and Orrenius, S. (1994) Intracellular Ca2/ signals activate apoptosis in thymocytes: Studies using the Ca2/ ATPase inhibitor thapsigargin. Exp. Cell Res. 212, 84–92. 41. Levick, V., Coffey, H., and D’Mello, S. R. (1995) Opposing effects of thapsigargin on the survival of developing cerebellar granule neurons in culture. Brain Res. 676, 325–335. 42. Kaneko, Y. and Tsukamoto, A. (1994) Thapsigargin-induced persistent intracellular calcium pool depletion and apoptosis in human hepatoma cells. Cancer Lett. 79, 147–155. 43. Choi, M. S., Boise, L. H., Gottschalk, A. R., Quintans, J., and Thompson, C. B. (1995) The role of BCL-XL in CD40-mediated rescue from anti-mu-induced apoptosis in WEHI-231 B lymphoma cells. Eur. J. Immunol. 25, 1352–1357. 44. Liu, J., J. D., Farmer, J., Lane, W. S., Friedman, J., Weissman, I., and Schreiber, S. L. (1991) Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66, 807–815. 45. Makrigiannis, A. P., Blay, J., and Hoskin, D. W. (1994) Cyclosporin A inhibits 2-chloroadenosine-induced DNA cleavage in mouse thymocytes. Int. J. Immunopharmacol. 16, 995– 1001. 46. Bonnefoy-Berard, N., Genestier, L., Flacher, M., and Revillard, J. P. (1994) The phosphoprotein phosphatase calcineurin controls calcium-dependent apoptosis in B cell lines. Eur. J. Immunol. 24, 325–329. 47. Amendola, A., Lombardi, G., Oliverio, S., Colizzi, V., and Piacentini, M. (1994) HIV-1 gp120-dependent induction of apoptosis in antigen-specific human T cell clones is characterized by ‘tissue’ transglutaminase expression and prevented by cyclosporin A. FEBS Lett. 339, 258–264. 48. Anel, A., Buferne, M., Boyer, C., Schmitt-Verhulst, A. M., and Golstein, P. (1994) T cell receptor-induced Fas ligand expression in cytotoxic T lymphocyte clones is blocked by protein tyrosine kinase inhibitors and cyclosporin A. Eur. J. Immunol. 24, 2469–2476. 49. Yazdanbakhsh, K., Choi, J. W., Li, Y., Lau, L. F., and Choi, Y. (1995) Cyclosporin A blocks apoptosis by inhibiting the DNA binding activity of the transcription factor Nur77. Proc. Natl. Acad. Sci. USA. 92, 437–441. 50. Shibasaki, F., Kondo, E., Akagi, E., and McKeon, F. (1997) Suppresion of signaling through NF-AT by interactions between calcineurin and BCL-2. Nature 386, 728–731. 51. Squier, M. K. T., Miller, A. C. K., Malkinson, A. M., and Cohen, J. J. (1994) Calpain activation in apoptosis. J. Cell Physiol. 159, 229–237. 52. Nath, R., Raser, K. J., Stafford, D., Hajimohammadreza, I., Posner, A., Allen, H., Talanian, R. V., Yuen, P., Gilbertsen, R. B., and Wang, K. K. (1996) Non-erythroid alpha-spectrin breakdown by calpain and interleukin 1 beta converting enzyme-like protease(s) in apoptotic cells: Contributory roles of both protease families in neuronal apoptosis. Biochem. J. 319, 683–690. 53. Vanags, D. M., Porn-Ares, M. I., Coppola, S., Burgess, D. H., and Orrenius, S. (1996) Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis. J. Biol. Chem. 271, 31075–31085. 54. Jordan, J., Galindo, M. F., and Miller, R. J. (1997) Role of calpain- and interleukin-1 beta converting enzyme-like proteases in the beta-amyloid-induced death of rat hippocampal neurons in culture. J. Neurochem. 68, 1612–1621. 55. Squier, M. K. T., and Cohen, J. J. (1997) Calpain, an upstream regulator of thymocyte apoptosis. J. Immunol. 158, 3690–3697. 56. Clawson, G. A., Norbeck, L. L., Hatem, C. L., Rhodes, C., Amiri, P., McKerrow, J. H., Patierno, S. R., and Fiskum, G. (1992)

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

Ca2/-regulated serine protease associated with the nuclear scaffold. Cell Growth & Diff. 3, 827–838. Tokes, Z. A., and Clawson, G. A. (1989) Proteolytic activity associated with the nuclear scaffold. The effect of self-digestion on lamins. J. Biol. Chem. 264, 15059–15065. Zhivotovsky, B., Gahm, A., Andaracrona, M., Nicotera, P., and Orrenius, S. (1995) Multiple proteases are involved in thymocyte apoptosis. Exp. Cell Res. 221, 404–412. McConkey, D. J. (1996) Calcium-dependent, interleukin 1bconverting enzyme (ICE) inhibitor-insensitive degradation of lamin B1 and DNA fragmentation in isolated thymocyte nuclei. J. Biol. Chem. 271, 22398–22406. Zhivotovsky, B., Gahm, A., and Orrenius, S. (1997) Two different proteases are involved in the proteolysis of lamin during apoptosis. Biochem. Biophys. Res. Comm. 233, 96–101. Chandra, J., Gilbreath, J., Freireich, E. J., Kliche, K. O., Andreeff, M., Keating, M., and McConkey, D. J. (1997) Protease activation is required for glucocorticoid-induced apoptosis in chronic lymphocytic leukemic lymphocytes. Blood in press. Lazebnik, Y. A., Takahashi, A., Moir, R. D., Goldman, R. D., Poirier, G. G., Kaufmann, S. H., and Earnshaw, W. C. (1995) Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc. Natl. Acad. Sci. USA. 92, 9042–9046. Schwartz, L. M., Smith, S. W., Jones, M. E. E., and Osborne, B. A. (1993) Do all programmed cell deaths occur via apoptosis? Proc. Natl. Acad. Sci. USA. 90, 980–984. Grimm, L. M., Goldberg, A. L., Poirier, G. G., Schwartz, L. M., and Osborne, B. A. (1996) Proteasomes play an essential role in thymocyte apoptosis. EMBO J. 15, 3835–3844. Sadoul, R., Fernandez, P. A., Quiquerez, A. L., Martinou, I., Maki, M., Schroter, M., Becherer, J. D., Irmler, M., Tschopp, J., and Martinou, J. C. (1996) Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons. EMBO J. 15, 3845–3852. Hewish, D. R. and Burgoyne, L. A. (1973) Chromatin sub-structure. The digestion of chromatin DNA at regularly spaced sites by a nuclear deoxyribonuclease. Biochem. Biophys. Res. Comm. 52, 504–510. Vanderbilt, J. N., Bloom, K. S., and Anderson, J. N. (1982) Endogenous nuclease: Properties and effects on transcribed genes in chromatin. J. Biol. Chem. 257, 13009–13017. Cohen, J. J., and Duke, R. C. (1984) Glucocorticoid activation of a calcium-dependent endonuclease in thymocyte nuclei leads to cell death. J. Immunol. 132, 38–42. Gaido, M. L., and Cidlowski, J. A. (1991) Identification, purification, and characterization of a calcium-dependent endonuclease (NUC 18) from apoptotic rat thymocytes. J. Biol. Chem. 266, 18580–18585. Montague, J. W., Gaido, M. L., Frye, C., and Cidlowski, J. A. (1994) A calcium-dependent nuclease from apoptotic rat thymocytes is homologous with cyclophilin. Recombinant cyclophilins A,B, and C have nuclease activity. J. Biol. Chem. 269, 18877– 18880. Peitsch, M. C., Polzar, B., Stephan, H., Crompton, T., MacDonald, H. R., Mannherz, H. G., and Tschopp, J. (1993) Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death). EMBO J. 12, 371–377. Ishida, R., Akiyoshi, H., and Takahashi, T. (1974) Isolation and purification of calcium and magnesium dependent endonuclease from rat liver nuclei. Biochem. Biophys. Res. Comm. 56, 703–708. Nikonova, L. V., Beletsky, I. P., and Umansky, S. R. (1993) Properties of some nuclear nucleases of rat thymocytes and

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their changes in radiation-induced apoptosis. Eur. J. Biochem. 215, 893–901. 74. Ribeiro, J. M., and Carson, D. A. (1993) Ca2//Mg2/-dependent endonuclease from human spleen: Purification, properties, and role in apoptosis. Biochemistry 32, 9129–9136. 75. Wyllie, A. H., Arends, M. J., Morris, R. G., Walker, S. W., and Evan, G. (1992) The apoptosis endonuclease and its regulation. Sem. Immunol. 4, 389–397. 76. Fesus, L., Thomazy, V., and Falus, A. (1987) Induction and activation of tissue transglutaminase during programmed cell death. FEBS Lett. 224, 104–108. 77. Fesus, L., Thomazy, V., Autuori, F., Ceru, M. P., Tarcsa, E., and Piacentini, M. (1989) Apoptotic hepatocytes become insoluble in detergents and chaotropic agents as a result of transglutaminase action. FEBS Lett. 245, 150–154. 78. Taresa, E., Kedei, N., Thomazy, V., and Fesus, L. (1992) An involucrin-like protein in hepatocytes serves as a substrate for tissue transglutaminase during apoptosis. J. Biol. Chem. 267, 25648–25651. 79. Fesus, L., Tarcsa, E., Kedei, N., Autuori, F., and Piacentini, M. (1991) Degradation of cells dying by apoptosis leads to accumulation of epsilon(gamma-glutamyl)lysine isodipeptide in culture fluid and blood. FEBS Lett. 284, 109–112. 80. Melino, G., Annicchiarico-Petruzzeli, M., Piredda, L., Candi, E., Gentile, V., Davies, P. J., and Piacentini, M. (1994) Tissue transglutaminase and apoptosis: Sense and antisense tranfection studies with human neuroblastoma cells. Mol. Cell. Biol. 14, 6584–6596. 81. Fadok, V. A., Savill, J. S., Haslett, C., Bratton, D. L., Doherty, D. E., Campbell, P. A., and Henson, P. M. (1992) Different populations of macrophages use either the vitronectin receptor or the phosphatidylserine receptor to recognize and remove apoptotic cells. J. Immunol. 149, 4029–4035. 82. Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L., and Henson, P. M. (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207–2216. 83. Verkleij, A. J., Zwaal, R. F., Roelofsen, B., Comfurius, P., Kastelijn, D., and van Deenen, L. L. (1973) The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy. Biochem. Biophys. Acta. 323, 178–193. 84. Zwaal, R. F., Roelofsen, B., Comfurius, P., and van Deenen, L. L. (1975) Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases. Biochem. Biophys. Acta. 406, 83–96. 85. Connor, J. and Schroit, A. J. (1990) Aminophospholipid translocation in erythrocytes: Evidence for the involvement of a specific transporter and an endofacial protein. Biochemistry 29, 37–43. 86. Bevers, E. M., Verahllen, P. F., Visser, A. J., Comfurius, P., and Zwaal, R. F. (1990) Bidirectional transbilayer lipid movement in human platelets as visualized by the fluorescent membrane probe 1[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene. Biochemistry 29, 5132–5137. 87. Sims, P. J., Wiedmer, T., Esmon, C. T., Weiss, H. J., and Shattil, S. J. (1989) Assembly of the platelet prothrombinase complex is linked to vesiculation of the platelet plasma membrane. Studies in Scott syndrome: An isolated defect in platelet procoagulant activity. J. Biol. Chem. 264, 17049–17057. 88. Fox, J. E., Austin, C. D., Reynolds, C. C., and Steffen, P. K. (1991) Evidence that agonist-induced activation of calpain causes the shedding of procoagulant-containing microvesicles from the membrane of aggregating platelets. J. Biol. Chem. 266, 13289–13295.

89. Sulpice, J. C., Zachowski, A., Devaux, P. F., and Giraud, F. (1994) Requirement for phosphatidylinositol 4,5-bisphosphate in the Ca2/-induced phospholipid redistribution in the human erythrocyte membrane. J. Biol. Chem. 269, 6347–6354. 90. Basse, F., Gaffet, P., Rendu, F., and Bienvenue, A. (1993) Translocation of spin-labeled phospholipids through the plasma membrane during thrombin- and ionophore A23187-induced platelet aggregation. Biochemistry 32, 2337–2344. 91. Bevers, E. M., Wiedmer, T., Comfurius, P., Zhao, J., Smeets, E. F., Schlegel, R. A., Schroit, A. J., Weiss, H. J., Williamson, P., Zwaal, R. F., and Sims, P. J. (1995) The complex of phosphatidylinositol 4,5-bisphosphate and calcium ions is not responsible for Ca2/-induced loss of phospholipid asymmetry in the human erythrocyte: A study of Scott syndrome, a disorder of calcium-induced phospholipid scrambling. Blood 86, 1983– 1991. 92. Williamson, P., Bevers, E. M., Smeets, E. F., Comfurius, P., Schlegel, R. A., and Zwaal, R. F. (1995) Continuous analysis of the mechanism of activated transbilayer lipid movement in platelets. Biochemistry 34, 10448–10455. 93. Verhoven, B., Schlegel, R. A., and Williamson, P. (1995) Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J. Exp. Med. 182, 1597– 1601. 94. Rosing, J., van Rijn, J. L., Bevers, E. M., van Dieijen, G., Comfurius, P., and Zwaal, R. F. (1985) The role of activated human platelets in prothrombin and factor X activation. Blood 65, 319– 332. 95. Schewe, M., Muller, P., Korte, T., and Herrmann, A. (1992) The role of phospholipid asymmetry in calcium phosphate-induced fusion of human erythrocytes. J. Biol. Chem. 267, 5910–5915. 96. Utsugi, T., Schroit, A. J., Connor, J., Bucana, C. D., and Fidler, I. J. (1991) Elevated expression of phosphatidylserine in the outer membrane leaflet of human tumor cells and recognition by activated human blood monocytes. Cancer Res. 51, 3062– 3066. 97. Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssiere, J. L., Petit, P. X., and Kroemer, G. (1995) Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J. Exp. Med. 181, 1661– 1672. 98. Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsch, T., Susin, S. A., Petit, P. X., Mignotte, B., and Kroemer, G. (1995) Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J. Exp. Med. 182, 367– 377. 99. Petit, P. X., Lecoeur, H., Zorn, E., Dauguet, C., Mignotte, B., and Gougeon, M. L. (1995) Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J. Cell Biol. 130, 157–167. 100. Hennet, T., Richter, C., and Peterhans, E. (1993) Tumour necrosis factor-alpha induces superoxide anion production in mitochondria of L929 cells. Biochem. J. 289, 587–592. 101. Lam, M., Dubyak, G., Chen, L., Nunez, G., Miesfeld, R. L., and Distelhorst, C. W. (1994) Evidence that bcl-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2/ fluxes. Proc. Natl. Acad. Sci. USA. 91. 102. Marin, M. C., Fernandez, A., Bick, R. J., Brisbay, S., Buja, M., Snuggs, M., McConkey, D. J., Eschenbach, A. C. V., Keating, M. J., and McDonnell, T. J. (1996) Apoptosis suppression by Bcl-2 is correlated with the regulation of nuclear and cytosolic Ca2/. Oncogene 12, 2259–2266. 103. Muchmore, S. W., Sattler, M., Liang, H., Meadows, R. P., Harlan, J. E., Yoon, H. S., Nettesheim, D., Chang, B. S., Thompson, C. B., Wong, S. L., Ng, S. C., and Fesik, S. W. (1996) X-ray and

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NMR structure of human BCL-XL, an inhibitor of programmed cell death. Nature 381, 335–341. 104. Antonsson, B., Conti, F., Ciavatta, A., Montessuit, S., Lewis, S., Martinou, I., Bernasconi, L., Bernard, A., Mermond, J., Mazzei, G., Maundrell, K., Gambale, F., Sadoul, R., and Martinou, J. C. (1997) Inhibition of BAX channel-forming activity by BCL2. Science 277, 370–372.

105. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T. I., Jones, D. P., and Wang, X. (1997) Prevention of apoptosis by bcl-2: Release of cytochrome c from mitochondria blocked. Science 275, 1129–1132. 106. Kluck, R. M., Bossy-Wetzel, E., Green, D. R., and Newmeyer, D. D. (1997) The release of cytochrome c from mitochondria: A primary site for bcl-2 regulation of apoptosis’. Science 275, 1132–1136.

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