INVITED REVIEW
Physiology of apoptosis

E. Gulbins, A. Jekle, K. Ferlinz, H. Grassmé, and F. Lang

Department of Physiology, University of Tuebingen, 72076 Tuebingen, Germany


    ABSTRACT
TOP
ABSTRACT
APOPTOTIC CELL DEATH
SIGNALING IN APOPTOSIS
ION CHANNELS IN APOPTOSIS
CONCLUSIONS
REFERENCES

Ion fluxes and volume changes of the whole cell as well as of organelles belong to the hallmarks of apoptosis; however, the molecular mechanism regulating these changes is only poorly characterized. Several ion channels in the plasma membrane, in particular the N-type K+ channel, the chloride channel cystic fibrosis conductance regulator, and an outward rectifying chloride channel, as well as the mitochondrial permeability transition pore, have been implicated to be involved in signal transduction cascades regulating apoptosis. Furthermore, Bcl-2-like proteins have been suggested to function, at least in part, as ion channels, because they display some homology to bacterial pore-forming toxins. In contrast to the demonstration of the involvement of these different ion channels in apoptosis, the molecular consequences regulated by these ion channels, and finally triggering apoptosis, are almost completely unknown.

CD95; ion channels; signaling; cell death; ceramide; sphingomyelinase


    APOPTOTIC CELL DEATH
TOP
ABSTRACT
APOPTOTIC CELL DEATH
SIGNALING IN APOPTOSIS
ION CHANNELS IN APOPTOSIS
CONCLUSIONS
REFERENCES

APOPTOSIS, OR PROGRAMMED CELL death, is an evolutionary, highly conserved, biological process requiring the regulated activation of several signaling cascades, which finally result in typical biochemical and morphological alterations of the cell. Multicellular organisms constantly delete and renew cells to maintain their homeostasis. Cellular turnover is a highly ordered and controlled event, being the combined result of cellular proliferation and cell death (34, 31). Death of cells in the normal turnover of tissues permits the removal of cells with genetic damage, those with improper developmental changes, or those that are produced in excess (34, 71, 105). A good example for such a function is provided by the CD95 receptor, which seems to predominantly regulate the peripheral immune response (94). Thus mutations in the CD95 receptor or its ligand result in the phenotype of either lpr and gld mice, characterized by lymphadenopathy, lymphoaccumulation, and autoimmune organ failure (72).

Changes typical for apoptosis include condensation of the cell and of the nuclei, DNA fragmentation into nucleosomal units of 200-bp fragments, chromatin condensation, generation of evoluted membrane segments (zeiosis), formation of apoptotic bodies, cellular shrinkage, and disintegration of mitochondria (52).

Apoptosis can be induced by many physiological and pathophysiological stimuli, including specific receptor molecules, e.g the CD95, Trail, or the tumor necrosis factor (TNF) receptor, but also by many stress forms, e.g., growth factor withdrawal, irradiation, ultraviolet light, heat shock, cytotoxic drugs, H2O2, ceramide treatment, bacteria, toxins, or viruses (2, 7, 13, 26, 44, 48, 74, 80, 95, 97).

Two examples will be discussed in more detail: the withdrawal of growth factors, e.g., interleukin-2 or nerve growth factor (NGF), results in the death of cells requiring these growth factors for proliferation or survival (2, 80). Thus these cells need continuous support to survive, and one can speculate that this continuous stimulus suppresses a preexistent death-signaling pathway in the cell. In contrast, triggering some receptors, e.g., the CD95 or TNF receptor, can actively initiate a previously inactive signaling or death program, finally resulting in apoptosis (48, 75).

Apoptotic cell death is distinguished from necrotic cell death in the morphological terms described above. Additionally, necrotic cell death is a passive process due to an acute cellular injury, whereas apoptotic cell death is an active process that requires participation of the dying cell and changes in cellular biochemistry.

Because apoptosis does not result in the release of intracellular material into the extracellular space, it usually does not result in an inflammatory response, whereas necrosis leads to cell disintegration and the induction of an unspecific and/or specific immune response.

Because that many stimuli include apoptosis, it is not surprising that defects in apoptotic cell death contribute to a number of human diseases.

First, in contrast to normal cells, which are deleted by apoptosis in the absence of growth factors and/or when present in an abnormal cellular environment, tumor cells develop independently from growth factors. This promotes their long-term survival and finally the development of a clinically manifest tumor. Proteins conferring the resistance to apoptotic cell death very often belong to the bcl-2 gene family (43). The exact molecular mechanisms of these proteins protecting cells from apoptosis are incompletely understood and will be discussed below.

Second, inactivating mutations of the CD95 receptor lead to the autoimmune lymphoproliferative syndrome in humans (30), which is homologous to the spontaneous mouse mutants lpr and gld carrying autosomal recessive mutations of the CD95 receptor or CD95 ligand, respectively. Humans suffer from lymphadenopathy, urticarial rashes, hemolytic anemia, thrombocytopenia, neutropenia, autoantibodies, and expansion of T cell antigen receptor (TCR)/CD3+, CD4-/8- lymphocytes, whereas mice develop a systemic lupus erythematosis-like syndrome and, consequently, glomerulonephritis. Additionally, lpr and gld mice exhibit a severe lymphoadenopathy due to accumulation of large numbers of CD4-/8-, TCR/CD3+ T cells in peripheral lymph nodes.

Third, cytotoxic T cells are able to kill virus-infected host cells by several mechanisms; i.e., they are able to express CD95 ligand and cross-link CD95 receptors on the target cell surface, resulting in apoptosis of the cell, or they secrete proteases that internally activate target cell apoptosis (5). In response to these defense mechanisms, several viruses obviously developed inhibitors of the apoptotic process to abolish these host defenses. These genes encode, e.g., the E1B protein of adenovirus, the BHRF1 of Epstein-Barr virus, and the LMW5-HL protein of African swine fever virus or the E10 protein of equine herpes virus 2 (16, 41, 73, 82, 102). These proteins display strong sequence similarity to bcl-2 and probably function in a very similar manner as Bcl-2 to protect cells from apoptosis. A distinct mechanism is demonstrated by the poxvirus gene crmA, which functions as a protease inhibitor, blocking the activity of some caspases, proteases that are absolutely crucial for the induction of apoptosis in many systems (83). These proteases are also targeted by viral FLICE-inhibitory proteins, which also prevent the apoptosis of the virus-infected cell (104).

Fourth, several studies suggest that depletion of CD4+ T cells in human immunodeficiency virus (HIV)-infected individuals involves the CD95 receptor. These data include the findings that CD95 receptor expression is elevated in HIV-infected individuals; T cells transformed by HIV are more susceptible to CD95-induced cell death; and stimulation of human or murine T cells with antibodies to CD4, a HIV receptor, induces increased CD95 expression and correlates with increased apoptotic cell death (42, 76, 77). Whether these findings also apply for the in vivo infection situation is still unclear.

Fifth, many data provide strong evidence that apoptosis plays a highly relevant role in human degenerative diseases, in particular neurodegenerative disorders, or in the tissue damage during or after hypoxia (92).

These examples are not complete and simply illustrate that apoptosis is emerging as an important parameter in many pathophysiological states. Understanding the apoptotic process, which appears as complex and highly regulated as that of proliferation, may yield valuable information in treatment of these diseases.


    SIGNALING IN APOPTOSIS
TOP
ABSTRACT
APOPTOTIC CELL DEATH
SIGNALING IN APOPTOSIS
ION CHANNELS IN APOPTOSIS
CONCLUSIONS
REFERENCES

In this section we will summarize some of the most important signaling events involved in apoptosis besides ion channels, which are discussed in the following section. However, it is impossible in this review focusing on ion channels to provide a complete picture of all signaling events involved in apoptosis. Almost all apoptotic stimuli induce the activation of some specific proteases, called caspases, which form a whole family of different cysteine-proteases cleaving target proteins at asparagine residues. Caspases have been divided into two groups, the initiator and the executor caspases, depending on their functional place in the apoptotic signaling cascade. A typical example for initiator caspases is caspase 8, which is rapidly activated by several receptors, in particular the CD95 or TNF receptor (9, 70). The CD95 receptor, a 48-kDa glycoprotein with a single membrane-spanning region, exhibits some sequence homology to TNF receptor-1 (p55) and -2 (p75), NGF receptors, CD40, OX40, CD27, and CD30, comprising the NGF/TNF receptor family (7). The intracellular part of CD95 displays a conserved domain with ~30% identity to the TNF receptor (101). This short part of the molecule encompassing about 70-80 amino acid residues was termed the "death domain" (101), because a deletion or mutations of this domain render the CD95 or TNF receptor inactive and prevent downstream signaling and programmed cell death. A typical feature of the death domain is the association with other death domains of other proteins or even the death domain of other CD95 receptor molecules (21, 84, 96). Thus the CD95 receptor is able to self-associate via the death domain. Additional proteins bearing a death domain-like region have been isolated by using the two-hybrid screening and include MORT1/FADD, TRADD, and RIP (9, 21, 70, 84, 96).

The present model of early CD95 receptor signaling predicts that, on trimerization or oligomerization of the CD95 receptor, FADD binds to the death domain of the CD95 receptor (9, 21, 70). FADD, in turn, binds to caspase 8 (FLICE/MACH-1) (9, 70), which contains a protease domain. The molecular mechanisms of caspase 8 activation are not well known, and it might be possible that ligation and trimerization of the CD95 receptor followed by the binding of caspase 8 to FADD induces a conformational change of caspase 8, resulting in its activation (Fig. 1). Caspase 8 then transmits the activation signal to other cellular proteins; however, most of the direct substrates are still unknown. One of the candidates for a caspase 8 substrate transferring the death signal to signal transduction elements further downstream is Bid (109). Bid belongs to the Bcl-2 protein family, and cleavage of Bid by caspase 8 triggers its translocation to the mitochondrium (35). Truncated Bid is a very potent inductor of apoptosis; however, the molecular details are unknown (35, 109).


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Fig. 1.   On trimerization of CD95, a caspase 8-regulated release of ceramide promotes the formation of membrane rafts, which provide the basis for efficient signaling of CD95. Those signaling events include a direct activation of caspase 3 via caspase 8 or an alteration of mitochondrial functions via still unknown intermediates. We assume that the ion channel in the cell membrane may predominantly function in the latter pathway. Further intermediates of this signaling pathway very likely include Bcl-2 like proteins such as Bid, Bim, or Bad and several ion channels in the mitochondrium itself. The alteration of the mitochondrium finally results in a release of cytochrome c and an activation of caspase 3, which induces the execution phase of cellular apoptosis. N-K+, N-type K+ channel; ORCC, outward rectifying chloride channel; CRAC, Ca2+-release-Ca2+ entry; PTP, permeability transition pore.

The activation of caspase 8 results in alterations of the mitochondrium (discussed in more detail in the following paragraph), which culminate in the release of cytochrome c from the mitochondrium. On release of cytochrome c into the cytosol, it forms a complex with caspase 9 and dATP, which finally seems to be able to stimulate the activity of caspase 3 (for review, see Ref. 55).

However, depending on the cell type it also seems to be possible that caspase 8 is directly stimulating these downstream events without involvement of the mitochondrium (87).

Caspase 3 is a typical member of the executor caspases and is able to cleave many cellular substrates, resulting in the typical features of apoptosis. The activation of caspase 3 is triggered by the limited proteolysis of the protein by other caspases such as caspase 9 or caspase 8.

Next, several groups provide an important function of sphingomyelinases for apoptosis (23, 36, 63). At least three different sphingomyelinases can be discriminated by their optimal pH: acid, neutral, and alkaline sphingomyelinases. In particular, acid sphingomyelinase has been implicated to be essential for apoptosis. This cellular glycoprotein seems to be directed either to acidic compartments to contribute to lysosomal sphingomyelin turnover or, as recently shown by Marathe et al. (63) and Schissel et al. (88) into secretory vesicles, which enable the protein to be secreted into the extracellular space. Secretory sphingomyelinase is derived from the same gene as acid sphingomyelinase and, besides a limited constitutive secretion rate, is strongly secreted on stimulation of macrophages or endothelial cells with inflammatory cytokines like interleukin-1beta (63). Interestingly, lysosomal acid sphingomyelinase and secretory sphingomyelinase are differentially processed at the NH2 terminus and, furthermore, display different modification by N-linked carbohydrate chains (88, 89). These differences very likely determine the route of the enzyme through the cellular compartments.

Because the activity of the enzyme can be modified by other lipids and, furthermore, displays ~5-10% of maximum activity at neutral pH (89), it is very likely that this extracellular form of acid sphingomyelinase is enzymatically functional.

Besides its crucial function in membrane turnover, acid sphingomyelinase has been shown by several groups to participate in receptor signaling, e.g., after stimulation via interleukin-1, TNF, CD95 receptor, or CD28, resulting in sphingomyelin hydrolysis and ceramide generation (15, 17, 23, 36, 91, 103). Furthermore, acid sphingomyelinase is activated by many forms of stress, in particular gamma and ultraviolet irradiation or cellular treatment with cytostatic drugs (39, 85). Most of these stimuli seem to activate acid sphingomyelinase within minutes after receptor stimulation, with a relatively low magnitude of activation ranging between 100 and 300%. However, because it is very likely that only a minor portion of cellular acid sphingomyelinase and not the majority of the lysosomal enzyme participates in signal transduction, it is possible that the biological effective, locally restricted enzyme activity is much higher. Recent genetic studies employing acid sphingomyelinase-knockout mice or lymphocytes from Niemann-Pick-disease type A patients, who suffer from an inborne defect of acid sphingomyelinase, confirmed the dominant role of acid sphingomyelinase in the cellular response to irradiation, ultraviolet light, or even after treatment with lipopolysaccharide (39, 40, 85).

However, an essential function of acid sphingomyelinase in CD95 and TNF-alpha receptor-triggered apoptosis has been recently questioned by a study from Cock et al. (25). A careful comparison of the data provided by this group suggests that the requirement of acid sphingomyelinase for receptor-mediated signaling and, thus apoptosis, critically depends on the amount of agonistic antibody applied. Preliminary experimental data from our group analyzing CD95 signaling strongly support this observation and suggest that low concentrations of receptor ligand fail to transmit the apoptotic signal in acid sphingomyelinase-deficient cells, whereas higher concentrations are effective independently of acid sphingomyelinase activity or expression. Because acid sphingomyelinase is not only activated by apoptotic stimuli, but also by the CD28 receptor (15), which induces cell proliferation or differentiation rather than apoptosis, the function of acid sphingomyelinase may not be restricted to the induction of apoptosis.

Thus an intriguing functional concept for the function of acid sphingomyelinase in receptor signaling activation suggests that the enzyme modifies membrane fluidity by formation of ceramide microdomains. Those structural alterations of the membrane morphology may then allow rapid and efficient signaling inside the cell, explaining the function of the enzyme in that many receptor systems. In this context acid sphingomyelinase would not function as a signaling molecule per se but rather provide the right environment for the receptor to initiate intracellular signal transduction either by interacting with other signaling molecules present in those ceramide-rich rafts or by simple close proximity and high density of the receptor and intracellular associating proteins.

In addition to acid sphingomyelinase several groups demonstrated an activation of neutral sphingomyelinase by the TNF-alpha or CD95 receptor. Recent studies from Hannun et al. (61) characterized the enzyme as an Mg2+ (Mn2+)-dependent integral transmembrane protein (61). The optimal enzymatic activity was observed at pH 7.5 and was dependent on the presence of acid phospholipid phosphatidylserine, whereas increase in glutathione concentration effectively blocked the enzyme (61). Neutral sphingomyelinases have been implicated in the regulation of cell differentiation, growth, apoptosis, and aging (53). Besides its activation on TNF-alpha (110) and CD95 receptor (24) ligation, the enzyme is also stimulated by neurotrophic factors (27), CD40 ligand (54), L-selectin (18), daunorubicine (13), dexamethasone (81), beta -D-cytosine arabinoside (98), and cell cycle arrest by serum deprivation or cell senescence (48). Thus it is very likely that ceramide released by neutral sphingomyelinase is also not a pure inductor of apoptosis but rather fulfills different functions depending on the context in which the lipid is acting in the cell.

A newly identified human alkaline sphingomyelinase from bile displayed an optimum activity at pH 9.0 and does not seem to participate in signal transduction events (28).

The complexity of these different enzymes, their distinct locations, and their different activation patterns are matched by a similar diversity of cellular effects triggered by the product of all sphingomyelinases, ceramide. In particular, ceramide has been shown to directly or indirectly interact with the kinase suppressor of Ras (KSR; identical to ceramide-activated protein kinase) (112), a ceramide-activated protein phosphatase (31), protein kinase Czeta (62, 68), and Raf-1 kinase (69). The KSR protein, a membrane-associated kinase with a substrate specificity for serine or threonine in proximity to proline, has been shown to play an important role in ceramide-mediated regulation of BAD, belonging to the Bcl-2-like protein family (3). The effect of ceramide on BAD was mediated by a pathway involving KSR, Ras, c-Raf-1, and mitogen-activated protein kinase kinase-1, finally causing prolonged inactivation of Akt, which normally inhibits BAD by phosphorylation. Reduction of Akt activity prevents phosphorylation of BAD, subsequently leading to BAD-mediated cell death.

Furthermore, ceramide has been demonstrated to activate Src-like tyrosine kinases, in particular p56Lck (38). However, the exact mechanism of ceramide-mediated tyrosine kinase activation has yet to be determined. The profound effect of ceramide on ion channels will be discussed in the next section.

Further molecules involved in apoptosis are protein tyrosine kinases, because inhibition of protein tyrosine kinases prevents or delays CD95 receptor-triggered programmed cell death (29, 90). An important function of Src-like tyrosine kinases for CD95-triggered apoptosis is also evidenced from experiments using Fyn- and Lyn-knockout mice, showing a deficiency of programmed cell death in peripheral B and T lymphocytes, by studies employing the Src-like tyrosine kinase inhibitors herbimycin A or tyrphostin B66, which almost completely prevented CD95 receptor-mediated programmed cell death, and, finally, by data displaying an association of the Src-like tyrosine-kinase p59fyn with the CD95 receptor (1, 29, 90).

Several other molecules have been shown to be involved in CD95 receptor-triggered apoptosis, in particular the small G protein p21Ras, and inhibition of Ras blocks CD95 receptor-induced programmed cell death in Jurkat T cells (36). Further molecules regulated on Fas receptor triggering include phospholipase A2 (24), nuclear factor-kappa B (79), Jun-N-terminal kinases (57), serine/threonine kinases (106), the tyrosine phosphatase FAP (86), or Rac proteins (19, 37).


    ION CHANNELS IN APOPTOSIS
TOP
ABSTRACT
APOPTOTIC CELL DEATH
SIGNALING IN APOPTOSIS
ION CHANNELS IN APOPTOSIS
CONCLUSIONS
REFERENCES

Several ion channels as well as transporters have been recently implicated to be involved and essential for apoptosis.

First, several studies show an inhibition of the N-type K+ channel (N-K+; Kv1.3) on CD95 receptor triggering or application of synthetic ceramides (38, 99). The N-type K+ channel is the predominant voltage-gated potassium channel in T lymphocytes; however, it is also expressed in neuronal cells. The above-mentioned studies directly determined the effect of CD95 receptor ligation and ceramides on the function of the N-K+ channel inactivation by whole-cell patch clamp experiments. CD95 receptor ligation or C6-ceramide application resulted in an ~50% inhibition of the current. Both stimuli reduced the open probability of the N-K+ channel but did not significantly alter the unitary current. The inhibition of the N-K+ channel was specific because neither the inactive compound dihydro-C2-ceramide nor other ceramide metabolites, sphingosine in particular, altered the channel function. Furthermore, preliminary studies employing B lymphocytes from a Niemann-Pick disease type A patient provide evidence that acid sphingomyelinase is required for CD95 receptor-triggered inhibition of the N-K+ channel. This suggests that the CD95 receptor employs acid sphingomyelinase and subsequently the released ceramide to inhibit the N-K+ channel.

The molecular mechanisms of CD95- or ceramide-mediated inhibition of N-K+ channels are not entirely clear, but the block of the potassium current correlates with tyrosine phosphorylation of the N-K+ channel. Supporting the evidence for a role of tyrosine phosphorylation of the channel protein in its inhibition, a genetic deficiency of p56lck, the major Src-like tyrosine kinase in Jurkat cells, or a pharmacological inhibition of Src-like tyrosine kinases prevents the inhibitory effect of CD95 receptor ligation or cellular ceramide treatment on the N-K+ channel. The increased tyrosine phosphorylation of the N-K+ channel protein correlated with a stimulation of Src-like tyrosine kinases such as p56lck on stimulation of the cells with C6-ceramide.

The molecular mechanism of tyrosine phosphorylation-mediated inhibition of N-type K+ channels is unknown. It might be possible that CD95 receptor ligation or ceramide treatment induces tyrosine phosphorylation of a highly conserved tyrosine in the H5 domain of the N-K+ channel. Tyrosine phosphorylation of this residue has been shown to inhibit the current of other voltage-gated K+ channels homologous to the N-K+ channel (47, 59). In addition, Holmes and co-workers (45) showed that phosphorylation of the N-K+ channel at tyrosine residue 449 suppresses the current. Whether this tyrosine residue is also phosphorylated on CD95 receptor ligation or ceramide triggering remains to be elucidated.

How does the N-type voltage-gated K+ channel regulate apoptosis? This channel has been shown to be responsible for the maintenance of the resting membrane potential in lymphocytes and, interestingly, margatoxin, which induces a strong depolarization of Jurkat cells, blocks cell proliferation (60). Therefore, it might be conceivable that alterations of the cell membrane potential are involved in activation of lymphocytes. In this concept, the resting membrane potential or a hyperpolarization would be required for cell proliferation, whereas depolarization of the membrane potential would be involved in the induction of apoptosis. However, the function of such a depolarized membrane potential in the apoptotic signaling machinery is unknown.

Furthermore, the N-K+ channel could also play a role as a scaffold protein in enabling the formation of large complexes of signaling molecules at the cell membrane. In this context it is interesting to note that another potassium channel, the Kv1.5 channel, has recently been shown to be associated with Src-like tyrosine kinases (46). The association was mediated by an interaction of the SH3 domain in the Src-kinase with a proline-rich domain in the channel protein.

An alternative hypothesis for the function of N-K+ channels would be that the channel is not only expressed in the cell membrane but also in cell organelles involved in apoptosis, in particular the nuclear or mitochondrial membranes. The inhibition of the channel protein in these organelles would have several distinct implications, in particular regulation of the mitochondrial membrane potential or the control of ion fluxes in the nucleus. The latter might be involved in the control of nuclear pores and, thus in the control of DNAses mediating DNA fragmentation during apoptosis.

The second group of ion channels obviously involved in apoptosis are several chloride channels. Thus it has been shown by Gottlieb et al. (33) that cells lacking the cystic fibrosis conductance regulator (CFTR) are resistant to cycloheximide- or etoposide-induced apoptosis. These studies correlated the genetic deficiency of functional CFTR with an insufficient cellular acidification during apoptosis, which has been suggested to be an important element of the death-signaling cascade (65). It was also shown that cellular acidification is downstream of caspases and Bcl-2. Thus the acidification of the cytoplasm might be involved in regulation of downstream or late events of the apoptotic signaling machinery. However, most enzymes involved in apoptosis do not seem to require an acid pH to be functionally active. Therefore, it might well be possible that CFTR may have additional functions in apoptosis, including the regulation of the ATP flux through the cell membrane or a function as an adapter protein for other signaling molecules involved in apoptosis.

The second chloride channel involved in apoptosis has been shown to function as an outward rectifying chloride channel (ORCC) (100). However, this channel has not been cloned as yet and, therefore, no molecular details of its role in apoptosis are known. The data showed a strong activation of the ORCC on CD95 receptor ligation in lymphocytes or after treatment of these cells with exogenous ceramides (100). These studies were performed with different patch- clamp techniques. This activation was dependent on the expression of the Src-like tyrosine kinase p56Lck in Jurkat T lymphocytes, suggesting that Src-like tyrosine kinases not only mediate inhibition of potassium channels but also activate chloride channels during apoptosis. The intimate role of Src-like tyrosine kinases for the activation of the ORCC was confirmed in excised patches incubated with recombinant p56Lck, being already sufficient to stimulate the ORCC. The function of the ORCC for apoptosis, however, is less well established mainly due to the lack of a genetic system to vigorously test the significance of the ion channel. However, pharmacological inhibition of the ORCC correlated with an up to 70% blockade of CD95-triggered death. Like with the N-K+ channel, the molecular mechanisms of ORCC involved in the apoptotic signaling machinery are completely unknown. It might be possible that the ORCC contributes to a change in the cellular pH or to the regulation of the cell membrane potential. Because the efflux of chloride should result in an alteration of the cell volume, it might be also conceivable that this channel contributes to the cell volume alteration observed in apoptosis. However, the time course of ORCC activation and cell shrinkage argues against such a possibility.

Third, P2x1 receptors might be involved in at least some forms of cellular apoptosis, because high extracellular ATP concentrations are able to trigger apoptosis in thymocytes (4, 22). P2x1 receptors are well-characterized receptors for ATP and function as nonselective cation channels. However, there is no definitive proof that ATP is killing cells via the activation of these channels proteins.

These and further, not yet characterized, ion channels might be also involved in the ion fluxes over the membrane described by Bortner and Cidlowski (10). This group described a strong efflux of potassium and sodium on induction of apoptosis by several stimuli, resulting in a strong decrease in the intracellular potassium concentration (12). A reduction in the intracellular potassium concentration seems to enhance the activation of caspases as well as DNases in the apoptotic signaling machinery. These authors also postulated that the decrease in the cell volume, which is observed concomitantly with apoptotic features, actively participates in the apoptosis-signaling process. Only cells with a reduced volume displayed an activation of caspases and DNA fragmentation, suggesting that the cell volume is not just a consequence of cell death but is also an important regulator of apoptotic signals (10). The notion of an important function of the cell volume for apoptosis is supported by findings that a twofold increase in the (extracellular) osmolarity triggers apoptosis (11, 64). However, several data argue against an important function of cell volume and ion fluxes over the membrane, which show that many apoptotic stimuli are able to trigger all apoptotic changes, including release of cytochrome c and fragmentation of nuclear DNA on addition to buffered cell lysates (14). Thus it might be possible that the observed ion fluxes and cell volume changes are facilitating apoptosis under normal conditions and are not absolutely essential for apoptosis but may rather function as enhancers. However, it might be also possible that the observed ion fluxes across the membrane do reflect very similar channel activities in cellular organelles, in particular the mitochondrium, the nucleus, or the endoplasmic reticulum, which are all intact even in the classic cell lysate experiments. In this scenario, the ion fluxes would be absolutely required for apoptosis.

Besides these ion channels, which are important in apoptosis, the CD95 receptor has been recently shown to inhibit the influx of Ca2+ via the cell membrane through Ca2+-release-Ca2+ entry (CRAC) channels (58). These channels are activated by mitogenic receptors, e.g., the TCR-CD3 complex in T lymphocytes, and mediate the long-term increase in intracellular Ca2+, which is important for the activation of transcription factors and finally gene transcription. These experiments showed that prestimulation of T lymphocytes via the CD95 receptor blocks the TCR/CD3-mediated activation of CRAC and the concomitant increase in intracellular Ca2+ concentration. This CD95-mediated inhibition of CRAC requires the expression of acid sphingomyelinase, and genetic deficiency of acid sphingomyelinase prevents the inhibitory effect of CD95 on CRAC. The important function of ceramide in this inhibitory effect of the CD95 receptor is also underlined by a very potent inhibition of CRAC by synthetic ceramides, whereas the inactive compound dihydro-C2-ceramide did not affect CRAC activity. However, in contrast to the ion channels described above, the inhibition of CRAC does not seem to be involved in apoptosis, because stimulation via CD95 alone does not alter the cellular Ca2+ concentration. The inhibition rather prevents a subsequent antigenic stimulation of this cell and, therefore, might be a mechanism to transiently block lymphocytes without deletion of the cell by apoptosis or even to anergize specific lymphocytes. The inhibition of TCR/CD3-mediated cell activation by the CD95 receptor-triggered blockade of CRAC might be also very important in the regulation of tumor growth. Many tumors express CD95 ligand on their surface or secrete the soluble CD95 ligand. The tumor-derived CD95 ligand may bind to the CD95 receptor of activated lymphocytes trying to attack the tumor. However, their stimulation via CD95 results, on one hand, in apoptosis of the immune cell and, on the other hand, in anergy of tumor-specific lymphocytes. This molecular mechanism finally results in the protection of the tumor against the immune system and, finally, in a growth advantage of the tumor.

However, some restrictions to this model may apply: superantigen-responding T cells rapidly expand and later decline after injection of mice with superantigen (32, 50, 51). The observed decline is not due to anergy or tolerance to the antigen but to a decrease in the number of cells expressing the appropriate TCRs capable of mounting a response. Thus the function of the CD95 receptor in this system is mainly to trigger apoptosis and not to induce an anergic state of activated T lymphocytes.

All ion channels discussed above have been demonstrated to be expressed in the plasma membrane.

Channel proteins expressed in mitochondria have been shown to be highly relevant for the regulation of apoptosis. The mitochondrium is one of the key players in most forms of apoptosis and undergoes several typical and irreversible alterations during the apoptotic process, finally leading to disruption of electron transport, ATP production, as well as the release of cytochrome c and apoptosis-inducing factor, which are absolutely critical for the activation of downstream caspases and DNAses, respectively (for review, see Ref. 55). Many of these mitochondrial alterations during apoptosis seem to be triggered by changes in the open probability of the permeability transition pore (PTP). The PTP seems to be composed of several proteins, including the voltage-dependent anion channel or porin, the adenine nucleotide translocator, cyclophilin, creatine kinase, and hexokinase (6, 8, 20, 113). In resting cells the PTP is closed, whereas apoptotic stimuli induce a great increase in the open probability of the PTP and inhibitors of the PTP have been shown to prevent at least some forms of apoptosis, indicating the significance of this protein complex for apoptosis (111). The opening of the PTP results in the release of small molecules up to ~1,500 Da from the mitochondria, uncoupling of the respiratory chain due to the free exchange of H+, reduction of the mitochondrial membrane potential Delta psi m, and swelling of the mitochondrial matrix. The molecular mechanisms regulating the opening of the PTP are not very well characterized. It has been shown that reactive oxygen intermediates and a swelling of the mitochondria are able to activate the PTP, which is prevented by interaction with antiapoptotic members of the Bcl-2-like protein family (93, 108). How are the cellular changes on the opening of the PTP connected with the release of cytochrome c and other molecules, finally triggering the death-signaling cascade? The swelling of the mitochondrial matrix may cause rupture of the outer mitochondrial membrane, enabling cytochrome c to leave the mitochondrium (78). However, a release of cytochrome c from the mitochondrium has been shown before or even without changes in the Delta psi m or PTP (108). Thus additional mechanisms seem to exist to trigger the release of cytochrome c from the mitochondrium. Mitochondria contain several systems for control of the concentration of K+ in the mitochondrial matrix, including the K+/H+ antiporter, some K+ channels, and the adenine nucleotide translocator. If these systems fail, the intramitochondrial K+ concentration increases, finally resulting in mitochondrial swelling and possibly rupture of the outer mitochondrial membrane.

In addition to mitochondrial swelling, the proapoptotic members of the Bcl-2-like protein family might be involved in the release of mitochondrial proteins during apoptosis. Interestingly, many of these proteins exhibit a pore-forming structure homologous to some bacterial toxins, in particular the diphtheria toxin and the colicins E1 and A (66, 67). This structure comprises five amphiphilic helices surrounding two hydrophobic helices. The BH1, BH2, and BH3 domains then form a hydrophobic pocket. In vitro experiments on lipid bilayers demonstrated that Bcl-XL can be inserted into these bilayers and forms a pH-sensitive and cation-selective channel (67). Furthermore, the selectivity varies among the different members of the Bcl-2 family due to the different BH1 and BH2 domains forming the pore. Thus Bcl-XL shows a conductance for K+ = Na+ and Ca2+ > Cl-, whereas the proapoptotic Bax shows a selectivity for Cl-. Even with structural data in mind, it is unclear whether Bcl-2-like proteins also function as ion channels or regulators of ion channels in vivo and, furthermore, how they mediate the release of cytochrome c from the mitochondrium.

Most of these events occur late in the cascade of mitochondrial alterations during apoptosis. Early events transmitting the signal from a receptor molecule or other stimuli onto the mitochondrium are almost completely unknown.

Besides classic ion channels, several transporters have been implicated to be involved in apoptosis. Thus it has been recently demonstrated in Jurkat T lymphocytes that cellular stimulation via the CD95 receptor triggers a marked release of cellular taurine after initiation of the death signal (56). This suggest that the cellular loss of taurine does not play a role in the early induction phase of cellular apoptosis, whereas the loss of taurine might be important for some apoptotic features in the later phase of the apoptotic process. The release of taurine could be involved, e.g., in late cellular shrinkage, a hallmark of apoptotic cells.

A second transporter possibly important for apoptosis seems to regulate the cellular concentration of glutathione (61, 107). Such a glutathione transporter has been shown to be activated early during apoptosis and, besides its effects on the cell volume, might regulate the function of neutral sphingomyelinase, because a reduction in cellular glutathione concentration strongly increases neutral sphingomyelinase activity (61). An increase or even normal physiological concentrations of glutathione very efficiently block the activity of this enzyme. Thus the activation of the glutathione transporter may finally result in the release of ceramide by neutral sphingomyelinase.


    CONCLUSIONS
TOP
ABSTRACT
APOPTOTIC CELL DEATH
SIGNALING IN APOPTOSIS
ION CHANNELS IN APOPTOSIS
CONCLUSIONS
REFERENCES

Ion channels comprise an emerging group of molecules playing an essential role in apoptosis. This includes ion channels in the plasma membrane as well as in certain cell organelles, in particular the mitochondrium. A participation of ion channels expressed in other organelles such as the nucleus or the endoplasmic reticulum has not been investigated as yet. Furthermore, the exact position and, in particular, regulation of ion channels during the apoptotic signaling cascade, is not very well characterized.

Apoptosis plays a very important role in many human diseases and is therefore an attractive therapeutic target. However, it has been difficult to manipulate apoptosis in vivo, because most drugs targeting intracellular signaling events involved in apoptosis are not very well membrane permeable. Therefore, ion channels with their extracellular domain might be very attractive targets for the development of drugs preventing or inducing apoptosis, because they can be easily reached and modified in the extracellular compartment.


    ACKNOWLEDGEMENTS

This work was supported in parts by Deutsche Forschungsgemeinschaft Grant Gu 335/2-2 and a grant from the German Cancer Research Foundation.


    FOOTNOTES

Address for reprint requests and other correspondence: E. Gulbins, Dept. of Physiology, Univ. of Tuebingen, Gmelinstrasse 5, 72076 Tuebingen, Germany (E-mail: erich.gulbins{at}uni-tuebingen.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
TOP
ABSTRACT
APOPTOTIC CELL DEATH
SIGNALING IN APOPTOSIS
ION CHANNELS IN APOPTOSIS
CONCLUSIONS
REFERENCES

1.   Atkinson, EA, Ostergaard H, Kane K, Pinkowski MJ, Caputo A, Olszowy MW, and Bleackley RC. A physical interaction between the cell death protein Fas and the tyrosine kinase p59fynT. J Biol Chem 271: 5968-5971, 1996[Abstract/Free Full Text].

2.   Baffy, G, Miyashita T, Williamson JR, and Reed JC. Apoptosis induced by withdrawal of 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, 1993[Abstract/Free Full Text].

3.   Basu, S, Bayoumy S, Zhang Y, Lozano J, and Kolesnick R. BAD enables ceramide to signal apoptosis via Ras and Raf-1. J Biol Chem 273: 30419-30426, 1998[Abstract/Free Full Text].

4.   Bean, BP. Pharmacology and electrophysiology of ATP-activated ion channels. Trends Pharmacol Sci 13: 87-90, 1992[ISI][Medline].

5.   Berke, G. The binding and lysis of target cells by cytotoxic lymphocytes: molecular and cellular aspects. Ann Rev Immunol 12: 735-773, 1994[ISI][Medline].

6.   Bernardi, P, and Petronilli V. The permeability transition pore as a mitochondrial calcium release channel: a critical appraisal. J Bioenerg Biomembr 28: 129-136, 1996.

7.   Beutler, B, and van Huffel C. Unraveling function in the TNF ligand and receptor families. Science 264: 667-668, 1994[ISI][Medline].

8.   Beutner, G, Rück A, Riede B, Welte W, and Brdiczka D. Complexes between kinases, mitochondrial porin, and adenylate translocator in rat brain resemble the permeability transition pore. FEBS Lett 396: 189-195, 1996[ISI][Medline].

9.   Boldin, MP, Goncharov TM, Goltsev YV, and Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85: 803-815, 1996[ISI][Medline].

10.   Bortner, CD, and Cidlowski JA. A necessary role for cell shrinkage in apoptosis. Biochem Pharmacol 56: 1549-1559, 1998[ISI][Medline].

11.   Bortner, CD, and Cidlowski JA. Absence of volume regulatory mechanisms contributes to the rapid activation of apoptosis in thymocytes. Am J Physiol Cell Physiol 271: C950-C961, 1996[Abstract/Free Full Text].

12.   Bortner, CD, Hughes FM, and Cidlowski JA. A primary role for K+ and Na+ efflux in the activation of apoptosis. J Biol Chem 272: 32436-32442, 1997[Abstract/Free Full Text].

13.   Bose, R, Verheij M, Haimovitz-Friedman A, Scotto K, Fuks Z, and Kolesnick R. Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 82: 405-414, 1995[ISI][Medline].

14.   Bossy-Wetzel, E, Newmeyer DD, and Green DR. Mitochondrial cytochrome C release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J 17: 37-49, 1998[Abstract/Free Full Text].

15.   Boucher, LM, Wiegmann K, Futterer A, Pfeffer K, Machleidt T, Schütze S, Mak TW, and Krönke M. CD28 signals through acidic sphingomyelinase. J Exp Med 181: 2059-2068, 1995[Abstract].

16.   Boyd, JM, Malstrom S, Subramanian T, Venkatesh LK, Schaeper U, Elangovan B, D'sa-Eipper C, and Chinnadurai G. Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins. Cell 79: 341-351, 1994[ISI][Medline].

17.   Brenner, B, Ferlinz K, Weller M, Grassmé H, Koppenhoefer U, Dichgans J, Sandhoff K, Lang F, and Gulbins E. Fas/CD95/APO-1 activates the acidic sphingomyelinase via ICE-like proteases. Cell Death Differ 5: 29-37, 1998[ISI][Medline].

18.   Brenner, B, Grassmé H, Müller C, Lang F, Speer CP, and Gulbins E. L-Selectin stimulates the neutral sphingomyelinase and induces release of ceramide. Exp Cell Res 243: 123-128, 1998[ISI][Medline].

19.   Brenner, B, Koppenhoefer U, Weinstock C, Linderkamp O, Lang F, and Gulbins E. Fas or ceramide induced apoptosis is mediated by a Rac 1 regulated activation of JNK/p38-Kinases and GADD 153. J Biol Chem 272: 22173-22181, 1997[Abstract/Free Full Text].

20.   Brustovetsky, N, and Klingenberg M. Mitochondrial ADP/ATP carrier can be reversibly converted into a large channel by Ca2+. Biochemistry 35: 8483-8488, 1996[ISI][Medline].

21.   Chinnaiyan, AM, O'Rourke K, Tewari M, and Dixit VM. FADD, a novel death domain-containing protein interacts with the death domain of Fas and initiates apoptosis. Cell 81: 505-512, 1995[ISI][Medline].

22.   Chvatchko, Y, Valera S, Aubry JP, Renno T, Buell G, and Bonnefoy JY. The involvement of an ATP-gated ion channel, P(2X1), in tymocyte apoptosis. Immunity 5: 275-283, 1996[ISI][Medline].

23.   Cifone, MG, De-Maria R, Roncaioli P, Rippo MR, Azuma M, Lanier LL, Santoni A, and Testi R. Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase. J Exp Med 180: 1547-1552, 1994[Abstract].

24.   Cifone, MG, Roncaioli P, de Maria R, Camarda G, Santoni A, Ruberti G, and Testi R. Multiple pathways originate at the Fas/APO-1 (CD95) receptor: sequential involvement of phosphatidylcholine-specific phospholipase C and acidic sphingomyelinase in the propagation of the apoptotic signal. EMBO J 14: 5859-5868, 1995[Abstract].

25.   Cock, JG, Tepper AD, de-Vries E, van-Blitterswijk WJ, and Borst J. CD95 (Fas/APO-1) induces ceramide formation and apoptosis in the absence of a functional acid sphingomyelinase. J Biol Chem 273: 7560-7565, 1998[Abstract/Free Full Text].

26.   Collins, M. Potential roles of apoptosis in viral pathogenesis. Am J Respir Crit Care Med 152: S20-24, 1995[ISI][Medline].

27.   Dobrowsky, RT, Jenkins GM, and Hannun YA. Neurotrophins induce sphingomyelin hydrolysis. Modulation by co-expression of p75NTR with Trk receptors. J Biol Chem 270: 22135-22142, 1995[Abstract/Free Full Text].

28.   Duan, RD, and Nilsson A. Purification of a newly identified alkaline sphingomyelinase in human bile and effects of bile salts and phosphatidylcholine on enzyme activity. Hepatology 26: 823-830, 1997[ISI][Medline].

29.   Eischen, CM, Dick CJ, and Leibson PJ. Tyrosine kinase activation provides an early and requisite signal for Fas-induced apoptosis. J Immunol 153: 1947-1953, 1994[Abstract/Free Full Text].

30.   Fisher, GH, Rosenberg FJ, Straus SE, Dale JK, Middleton LA, Lin AY, Strober W, Lenardo MJ, and Puck JM. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81: 935-946, 1995[ISI][Medline].

31.   Galadari, S, Kishikawa K, Kamibayashi C, Mumby MC, and Hannun YA. Purification and characterization of ceramide-activated protein phosphatases. Biochemistry 37: 11232-11238, 1998[ISI][Medline].

32.   Gonzalo, JA, Moreno de Alboran I, Ales-Martinez JE, Martinez C, and Kroemer G. Expansion and clonal deletion of peripheral T cells induced by bacterial superantigen is independent of the interleukin-2 pathway. Eur J Immunol 22: 1007-1011, 1992[ISI][Medline].

33.   Gottlieb, RA, and Dosanjh A. Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis. Proc Natl Acad Sci USA 93: 3587-3591, 1996[Abstract/Free Full Text].

34.   Green, DR, and Scott DW. Activation-induced apoptosis in lymphocytes. Curr Opin Immunol 6: 476-87, 1994[ISI][Medline].

35.   Gross, A, Yin XM, Wang K, Wei MC, Jockel J, Milliman C, Erdjument-Bromage H, Tempst P, and Korsmeyer SJ. Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. J Biol Chem 274: 1156-1163, 1999[Abstract/Free Full Text].

36.   Gulbins, E, Bissonette R, Mahboubi A, Nishioka W, Brunner T, Baier G, Baier-Bitterlich G, Byrd C, Lang F, Kolesnick R, Altman A, and Green D. Fas-induced apoptosis is mediated via a ceramide-initiated Ras signaling pathway. Immunity 2: 341-351, 1995[ISI][Medline].

37.   Gulbins, E, Coggeshall KM, Brenner B, Schlottmann K, Linderkamp O, and Lang F. Fas-induced apoptosis is mediated by activation of a Ras and Rac protein-regulated signaling pathway. J Biol Chem 271: 26389-26394, 1996[Abstract/Free Full Text].

38.   Gulbins, E, Szabo I, Baltzer K, and Lang F. Ceramide induced inhibition of T-lymphocyte voltage gated potassium channel is mediated by tyrosine kinases. Proc Natl Acad Sci USA 94: 7661-7666, 1997[Abstract/Free Full Text].

39.   Haimovitz-Friedman, A, Cordon-Cardo C, Bayoumy S, Garzotto M, McLoughlin M, Gallily R, Edwards CK, Schuchman EH, Fuks Z, and Kolesnick R. Liopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation. J Exp Med 186: 1831-1841, 1997[Abstract/Free Full Text].

40.   Haimovitz-Friedman, A, Kan CC, Ehleiter D, Persaud RS, McLoughlin M, Fuks Z, and Kolesnick RN. Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis. J Exp Med 180: 525-535, 1994[Abstract].

41.   Henderson, S, Huen D, Rowe M, Dawson C, Johnson G, and Rickinson A. Epstein-Barr virus-coded BHRF1 protein, a viral homologue of Bcl-2, protects human B cells from programmed cell death. Proc Natl Acad Sci USA 90: 8479-8483, 1993[Abstract/Free Full Text].

42.   Hiromatsu, KY, Aoki M, Makino M, Matsumoto Y, Mizuochi T, Gotoh Y, Nomoto K, Ogasawara J, Nagata S, and Yoshikai Y. Increased Fas antigen expression in murine retrovirus-induced immunodeficiency syndrome, MAIDS. Eur J Immunol 24: 2446-2451, 1994[ISI][Medline].

43.   Hockenbery, DM, Nunez G, Milliman C, Schreiber RD, and Korsmeyer SJ. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348: 334-336, 1990[ISI][Medline].

44.   Hockenbery, DM, Oltvai ZN, Yin XM, Milliman CL, and Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75: 241-251, 1993[ISI][Medline].

45.   Holmes, TC, Fadool DA, and Levitan IB. Tyrosine phosphorylation of the Kv1.3 potassium channel. J Neurosci 16: 1581-1590, 1996[Abstract].

46.   Holmes, TC, Fadool DA, Ren R, and Levitan IB. Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain. Science 274: 2089-2091, 1996[Abstract/Free Full Text].

47.   Huang, XY, Morielli AD, and Peralta EG. Tyrosine kinase-dependent suppression of a potassium channel by the G protein-coupled m1 muscarinic acetylcholine receptor. Cell 75: 1145-1156, 1993[ISI][Medline].

48.   Itoh, N, Yonehara S, Ishii A, Yonehara M, Mizushima S, Sameshima M, Hase A, Seto Y, and Nagata S. The polypeptide encoded by the cDNA for human cell surface antigen (Fas) can mediate apoptosis. Cell 66: 233-243, 1991[ISI][Medline].

49.   Jayadev, S, Liu B, Bielawska AE, Lee JY, Nazaire F, Pushkareva MY, Obeid LM, and Hannun YA. Role for ceramide in cell cycle arrest. J Biol Chem 270: 2047-2052, 1995[Abstract/Free Full Text].

50.   Jones, LA, Chin LT, Longo DL, and Kruisbeek AM. Peripheral clonal elimination of functional T cells. Science 250: 1726-1729, 1990[ISI][Medline].

51.   Kawabe, Y, and Ochi A. Programmed cell death and extrathymic reduction of Vbeta 8+ CD4+ T cells in mice tolerant to Staphylococcus aureus enterotoxin B. Nature 349: 245-248, 1991[ISI][Medline].

52.   Kerr, JFR, Wyllie AH, and Currie AR. Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 26: 239-257, 1972[ISI][Medline].

53.   Kolesnick, RN, and Krönke M. Regulation of ceramide production and apoptosis. Annu Rev Physiol 60: 643-665, 1998[ISI][Medline].

54.   Koppenhoefer, U, Brenner B, Lang F, and Gulbins E. The CD40-ligand stimulates T-lymphocytes via the neutral sphingomyelinase: a novel function of the CD40-ligand as signalling molecule. FEBS Lett 414: 444-448, 1997[ISI][Medline].

55.   Kroemer, G, Dallaporta B, and Resche-Rigon M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60: 619-642, 1998[ISI][Medline].

56.   Lang, F, Madlung J, Uhlemann AC, Risler T, and Gulbins E. Cellular taurine release triggered by stimulation of the Fas (CD95) receptor in Jurkat lymphocytes. Eur J Physiol 436: 377-383, 1998[ISI][Medline].

57.   Latinis, KM, and Koretzky GA. Fas ligation induces apoptosis and Jun kinase activation independently of CD45 and Lck in human T cells. Blood 87: 871-875, 1996[Abstract/Free Full Text].

58.   Lepple-Wienhues, A, Belka C, Laun T, Jekle A, Walter B, Wieland U, Welz M, Heil L, Kun J, Busch G, Weller M, Bamberg M, Gulbins E, and Lang F. Stimulation of CD95 Fas blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids. Proc Natl Acad Sci USA 96: 13795-13800, 1999[Abstract/Free Full Text].

59.   Lev, S, Moreno H, Martinez R, Canoll P, Peles E, Musacchio JM, Plowman GD, Rudy B, and Schlessinger J. Protein tyrosine PYK2 involved in Ca2+-induced regulation of ion channel and MAP kinase functions. Nature 376: 737-745, 1995[ISI][Medline].

60.   Lin, CS, Boltz RC, Blake JT, Nguyen M, Talento A, Fischer PA, Springer MS, Sigal NH, Slaughter RS, Garcia ML, Kaczorowski JG, and Koo GC. Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation. J Exp Med 177: 637-645, 1993[Abstract].

61.   Liu, B, Andrieu-Abadie N, Levade T, Zhang P, Obeid LM, and Hannun YA. Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death. J Biol Chem 273: 11313-11320, 1998[Abstract/Free Full Text].

62.   Lozano, J, Berra E, Municio MM, Diaz-Meco MT, Dominguez I, Sanz L, and Moscat J. Protein kinase C zeta isoform is critical for kappa B-dependent promoter activation by sphingomyelinase. J Biol Chem 69: 19200-19202, 1994.

63.   Marathe, S, Schissel SL, Yellin MJ, Beatini N, Mintzer R, Williams KJ, and Tabas I. Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase. Implications for early atherogenesis and ceramide-mediated cell signaling. J Biol Chem 273: 4081-4088, 1998[Abstract/Free Full Text].

64.   Matthews, CC, and Feldman EL. Insulin-like growth factor I rescues SH-SY5Y human neuroblastoma cells from hyperosmotic induced programmed cell death. J Cell Physiol 166: 323-331, 1996[ISI][Medline].

65.   Meisenholder, GW, Martin SJ, Green DR, Nordberg J, Babior BM, and Gottlieb RA. Events in apoptosis. Acidification is downstream of protease activation and BCL-2 protection. J Biol Chem 271: 16260-16262, 1996[Abstract/Free Full Text].

66.   Minn, AJ, Velez P, Schendel SL, Liang H, Muchmore SW, Fesik SW, Fill M, and Thompson CB. Bcl-xL forms an ion channel in synthetic lipid membranes. Nature 385: 353-357, 1997[ISI][Medline].

67.   Muchmore, SW, Sattler M, Liang H, Meadows RP, Harlan JE, Yoon HS, Nettesheim D, Chang BS, Thompson CB, Wong SL, Ng SL, and Fesik SW. X-ray and NMR structure of Bcl-xL, an inhibitor of programmed cell death. Nature 381: 335-341, 1996[ISI][Medline].

68.   Müller, G, Ayoub M, Storz P, Rennecke J, Fabbro D, and Pfizenmaier K. PKC zeta is a molecular switch in signal transduction of TNF-alpha, bifunctionally regulated by ceramide and arachidonic acid. EMBO J 4: 1961-1969, 1995.

69.   Müller, G, Storz P, Bourteele S, Doppler H, Pfizenmaier K, Mischak H, Philipp A, Kaiser C, and Kolch W. Regulation of Raf-1 kinase by TNF via its second messenger ceramide and cross-talk with mitogenic signalling. EMBO J 17: 732-742, 1998[Abstract/Free Full Text].

70.   Muzio, M, Chinnaiyan AM, Kischkel FC, O'Rourke K, Shevchenko A, Ni J, Scaffidi C, Bretz JD, Zhang M, Gentz R, Mann M, Krammer PH, Peter ME, and Dixit VM. FLICE, a novel FADD-homologous ICE/CED-3 like-protease, is recruited to the CD95 /Fas/Apo-I) death inducing signaling complex. Cell 85: 817-827, 1996[ISI][Medline].

71.   Nagata, S, and Golstein P. The Fas death factor. Science 267: 1449-1456, 1995[ISI][Medline].

72.   Nagata, S, and Suda T. Fas and Fas ligand: lpr and gld mutations. Immunol Today 16: 39-43, 1995[ISI][Medline].

73.   Neilan, JG, Lu Z, Afonso CL, Kutish GF, Sussman MD, and Rock DL. An African swine fever virus gene with similarity to the proto-oncogene bcl-2 and the Epstein-Barr virus gene BHRF1. J Virol 67: 4391-4394, 1993[Abstract].

74.   Obeid, LM, Linardic CM, Karolak LA, and Hannun YA. Programmed cell death induced by ceramide. Science 259: 1769-1771, 1993[ISI][Medline].

75.   Oehm, A, Behrmann I, Weih F, Pawlita M, Maier G, Klas C, Li WM, Richards S, Dhein J, Trauth BC, Ponstingl H, and Krammer PH. Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily: sequence identity with the Fas antigen. J Biol Chem 267: 10709-10715, 1992[Abstract/Free Full Text].

76.   Oyaizu, N, McCloskey TW, Coronesi M, Chirmule N, Kalyanaraman VS, and Pahwa S. Accelerated apoptosis in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus type-1 infected patients and in CD4 cross-linked PBMCs from normal individuals. Blood 82: 3392-3400, 1993[Abstract].

77.   Oyaizu, N, McCloskey TW, Than S, Hu R, Kalyanaraman VS, and Pahwa S. Cross-linking of CD4 molecules upregulates Fas antigen expression in lymphocytes by inducing interferon-gamma and tumor necrosis factor-alpha secretion. Blood 84: 2622-2631, 1994[Abstract/Free Full Text].

78.   Petit, PX, Susin SA, Zamzami N, Mignotte B, and Kroemer G. Mitochondria and programmed cell death: back to the future. FEBS Lett 396: 7-13, 1996[ISI][Medline].

79.   Ponton, A, Clement MV, and Stamenkovic I. The CD95 (Apo-1/Fas) receptor activates NF-kappa B independently of its cytotoxic function. J Biol Chem 271: 8991-8995, 1996[Abstract/Free Full Text].

80.   Raff, MC, Barres BA, Burne JF, Coles HS, Ishizaki Y, and Jacobson MD. Programmed cell death and the control of cell survival: lessons from the nervous system. Science 262: 695-700, 1993[ISI][Medline].

81.   Ramachandran, CK, Murray DK, and Nelson DH. Dexamethasone increases neutral sphingomyelinase activity and sphingosine levels in 3T3-L1 fibroblasts. Biochem Biophys Res Commun 167: 607-613, 1990[ISI][Medline].

82.   Rao, L, Debbas M, Sabbatini P, Hockenbery D, Korsmeyer S, and White E. The adenovirus E1A proteins induce apoptosis, which is inhibited by the E1B 19-kDa and Bcl-2 proteins. Proc Natl Acad Sci USA 89: 7742-7746, 1992[Abstract].

83.   Ray, CA, Black RA, Kronheim SR, Greenstreet TA, Sleath PR, Salvesen GS, and Pickup DJ. Viral inhibition of inflammation: cowpox virus encodes an inhibitor of the interleukin-1 beta converting enzyme. Cell 69: 597-604, 1992[ISI][Medline].

84.   Rothe, M, Wong SC, Henzel WJ, and Goeddel DV. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78: 681-692, 1994[ISI][Medline].

85.   Santana, P, Pena LA, Haimovitz-Friedman A, Martin S, Green D, McLoughlin M, Cordon-Cardo C, Schuchman EH, Fuks Z, and Kolesnick R. Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis. Cell 26: 189-199, 1996.

86.   Sato, T, Irie S, Kitada S, and Reed JC. FAP-1: a protein tyrosine phosphatase that associates with Fas. Science 268: 411-415, 1995[ISI][Medline].

87.   Scaffidi, C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH, and Peter ME. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17: 1675-1687, 1998[Abstract/Free Full Text].

88.   Schissel, SL, Jiang X, Tweedie-Hardman J, Jeong T, Camejo EH, Najib J, Rapp JH, Williams KJ, and Tabas I. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development. J Biol Chem 273: 2738-2746, 1998[Abstract/Free Full Text].

89.   Schissel, SL, Kessler GA, Schuchman EH, Williams KJ, and Tabas I. The cellular trafficking and zinc dependence of secretory and lysosomal sphingomyelinase, two products of the acid sphingomyelinase gene. J Biol Chem 273: 18250-18259, 1998[Abstract/Free Full Text].

90.   Schlottmann, K, Gulbins E, Lau S, and Coggeshall KM. Activation of and requirement for Src-family tyrosine kinases in Fas-induced apoptosis. J Leukoc Biol 60: 546-554, 1996[Abstract].

91.   Schütze, S, Potthoff K, Machleidt T, Berkovic D, Wiegmann K, and Krönke M. TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell 71: 765-776, 1992[ISI][Medline].

92.   Schulz, JB, Weller M, and Moskowitz MA. Caspases as treatment targets in stroke and neurodegenerative diseases. Ann Neurol 45: 421-429, 1999[ISI][Medline].

93.   Scorrano, L, Petronilli V, and Bernardi P. On the voltage dependence of the mitochondrial permeability transition pore. A critical appraisal. J Biol Chem 272: 12295-12299, 1997[Abstract/Free Full Text].

94.   Singer, GG, and Abbas AK. The Fas antigen is involved in peripheral but not thymic deletion of T lymphocytes in T cell receptor transgenic mice. Immunity 1: 365-371, 1994[ISI][Medline].

95.   Smith, CA, Farrah T, and Goodwin RG. The TNF-receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 76: 959-962, 1994[ISI][Medline].

96.   Stanger, BZ, Leder P, Lee TH, Kim E, and Seed B. RIP: a novel protein containing a death domain that interacts with Fas/Apo1 (CD95) in yeast and causes cell death. Cell 81: 513-523, 1995[ISI][Medline].

97.   Stevenson, MA, Pollock SS, Coleman CN, and Calderwood SK. X-irradiation, phorbol esters and H2O2 stimulate mitogen activated protein kinase activity in NIH-3T3-cells through the formation of reactive oxygen intermediates. Cancer Res 54: 12-15, 1994[Abstract].

98.   Strum, JC, Small GW, Pauig SB, and Daniel LW. 1-Beta-D-arabino-furanosylcytosine stimulates ceramide and diglyceride formation in HL-60 cells. J Biol Chem 269: 15493-15497, 1994[Abstract/Free Full Text].

99.   Szabo, I, Gulbins E, Zhang X, Apfel H, Barth P, Busch AE, Koppenhoefer U, Schlottmann K, Pongs O, and Lang F. Tyrosine phosphorylation dependent suppression of a voltage-gated K+ channel in T-lymphocytes upon Fas stimulation. J Biol Chem 271: 20465-20469, 1996[Abstract/Free Full Text].

100.   Szabo, I, Lepple-Wienhues A, Kaba N, Zoratti M, Gulbins E, and Lang F. Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis. Proc Natl Acad Sci USA 95: 6169-6174, 1998[Abstract/Free Full Text].

101.   Tartaglia, LA, Ayres TM, Wong GHW, and Goeddel DV. A novel domain within the 55 kd TNF receptor signals cell death. Cell 74: 845-853, 1993[ISI][Medline].

102.   Telford, EA, Watson MS, Aird HC, Perry J, and Davison AJ. The DNA sequence of equine herpes virus 2. J Mol Biol 249: 520-528, 1995[ISI][Medline].

103.   Tepper, CG, Jayadev S, Liu B, Bielawska A, Wolff R, Yonehara S, Hannun YA, and Seldin MF. Role for ceramide as an endogenous mediator of Fas-induced cytotoxicity. Proc Natl Acad Sci USA 92: 8443-8447, 1995[Abstract].

104.   Thome, M, Schneider P, Hofmann K, Fickenscher H, Meinl E, Neipel F, Mattmann C, Burns K, Bodmer JL, Schroter M, Scaffidi C, Krammer PH, Peter ME, and Tschopp J. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386: 517-521, 1997[ISI][Medline].

105.   Thompson, CB. Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456-1462, 1995[ISI][Medline].

106.   Tian, Q, Taupin J, Elledge S, Robertson M, and Anderson P. Fas-activated serine/threonine kinases (FAST) phosphorylates TIA-1 during Fas-mediated apoptosis. J Exp Med 182: 865-874, 1995[Abstract].

107.   Van den Dobbelstein, DJ, Nobel CSI, Schlegel J, Cotgreave IA, Orrenius S, and Slater AF. Rapid and specific efflux of reduced glutathione during apoptosis induced by anti-Fas/APO-1 antibody. J Biol Chem 271: 15420-15427, 1996[Abstract/Free Full Text].

108.   Van der Heiden, GM, Chandel NS, Williamson EK, Schumacker PT, and Thompson CB. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91: 627-637, 1997[ISI][Medline].

109.   Wang, K, Yin XM, Chao DT, Milliman CL, and Korsmeyer SJ. BID: a novel BH3 domain-only death agonist. Genes Dev 10: 2859-2869, 1996[Abstract].

110.   Wiegmann, K, Schutze S, Machleidt T, Witte D, and Krönke M. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78: 1005-1015, 1994[ISI][Medline].

111.   Zamzami, N, Marchetti P, Castedo M, Hirsch T, Susin SA, Masse B, and Kroemer G. Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis. FEBS Lett 384: 53-57, 1996[ISI][Medline].

112.   Zhang, Y, Yao B, Delikat S, Bayoumy S, Lin XH, Basu S, McGinley M, Chan-Hui PY, Lichenstein H, and Kolesnick R. Kinase suppressor of Ras is ceramide-activated protein kinase. Cell 89: 63-72, 1997[ISI][Medline].

113.   Zoratti, M, and Szabo I. The mitochondrial permeability transition. Biochim Biophys Acta 1241: 139-176, 1995[ISI][Medline].


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