Download Beyond apoptosis: nonapoptotic cell death in physiology and disease

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REVIEW / SYNTHÈSE
Beyond apoptosis: nonapoptotic cell death in
physiology and disease
Claudio A. Hetz, Vicente Torres, and Andrew F.G. Quest
Abstract: Apoptosis is a morphologically defined form of programmed cell death (PCD) that is mediated by the activation
of members of the caspase family. Analysis of death-receptor signaling in lymphocytes has revealed that caspase-dependent
signaling pathways are also linked to cell death by nonapoptotic mechanisms, indicating that apoptosis is not the only
form of PCD. Under physiological and pathological conditions, cells demonstrate a high degree of flexibility in cell-death
responses, as is reflected in the existence of a variety of mechanisms, including necrosis-like PCD, autophagy (or type
II PCD), and accidental necrosis. In this review, we discuss recent data suggesting that canonical apoptotic pathways,
including death-receptor signaling, control caspase-dependent and -independent cell-death pathways.
Key words: apoptosis, necrosis, nonapoptotic programmed cell death, death receptors, ceramides.
Résumé : L’apoptose est une forme de mort cellulaire programmée (MCP) définie morphologiquement qui est contrôlée
par l’activité des membres de la famille des caspases. L’analyse de la signalisation issue des récepteurs de mort chez
les lymphocytes a révélé que les voies signalétiques dépendantes des caspases étaient aussi liées à la mort cellulaire
causée par d’autres formes de mort cellulaire programmée (MCP). Sous des conditions physiologiques ou pathologiques,
les cellules font preuve d’un haut degré de flexibilité dans leurs réponses menant à la mort, comme le reflète l’existence
d’une variété de mécanismes incluant la MCP apparentée à la nécrose, l’autophagie (ou MCP de type II) et la nécrose
accidentelle. Dans cette revue, nous discutons des plus récents résultats suggérant que les sentiers apoptotiques fondamentaux, incluant la signalisation par les récepteurs de mort, contrôlent les sentiers de mort dépendants et indépendants
des capsases.
Mots clés : apoptose, nécrose, mort cellulaire programmée non-apoptotique, récepteurs de mort, céramides.
[Traduit par la Rédaction]
Hetz et al.
588
Apoptosis, nonapoptotic programmed cell
death, and accidental necrosis
In general, programmed cell death (PCD) is defined as an
active process that depends on the execution of a defined sequence of signaling events that lead to cell demise. Because
the precise nature of molecular events associated with various PCD pathways is not well understood, morphological
and biochemical criteria employed to define distinctive PCD
programs and their molecular pathways will be discussed in
this review.
Received 3 January 2005. Revision received 24 March 2005.
Accepted 13 April 2005. Published on the NRC Research
Press Web site at http://bcb.nrc.ca on 31 August 2005.
C.A. Hetz.1 Instituto de Ciencias Biomédicas, Universidad de
Chile, Santiago, Chile.
V. Torres and A.F.G. Quest.2 FONDAP Center for
Molecular Studies of the Cell (CEMC), Universidad de Chile,
Av. Independencia 1027, Santiago, Chile.
1
Present address: Dana-Farber Cancer Institute, Department of
Cancer Immunology and AIDS, Boston, MA 02115, USA.
2
Corresponding author (e-mail: [email protected]).
Biochem. Cell Biol. 83: 579–588 (2005)
Apoptosis is a particular morphological manifestation of
PCD (Kerr et al. 1972; Lockshin and Williams 1965), and
constitutes a highly conserved pathway that, in its basic
features, appears to operate in all metazoans. During embryonic development, apoptosis is essential for successful
organogenesis and participates in the control of cellular
populations (Vaux and Korsmeyer 1999). Apoptosis also
operates in adult organisms to maintain normal cellular homeostasis, which is particularly important with respect to
the development of disease in humans.
Early studies performed by Kerr et al. (1972) defined the
central morphological features of cells undergoing
apoptosis during development, such as cytoplasmic condensation, nuclear pyknosis, stage 2 chromatin condensation,
membrane blebbing, and generation of apoptotic bodies
that are normally eliminated by phagocytic cells in the surrounding tissue. In the past 15 years, additional markers of
apoptosis have been found, including DNA fragmentation,
phosphatidylserine exposure at the cell surface, and the
characterization of key molecular players, such as cysteine
proteases of the caspase family (Degterev et al. 2003),
many adaptor proteins, and members of the Bcl-2 family of
proteins (Danial and Korsmeyer 2004). In essence, pathways
doi: 10.1139/O05-065
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leading to apoptosis via either the extrinsic (death-receptor
mediated) or intrinsic (i.e., triggered by endogenous mechanisms related to cellular stress) pathway (Hengartner 2000;
Razik and Cidlowski 2002) are becoming increasingly appreciated as highly orchestrated macromolecular assembly
processes (Leyton and Quest 2002, 2004). Understanding
such processes better holds the promise of promoting or
preventing cell-death-related events and, hence, treating a
variety of human diseases (Li et al. 2004; Walensky et al.
2004).
Although apoptosis, a caspase-mediated process, is the
prevalent form of PCD employed to control cell viability
during development and to maintain homeostasis, recent data
indicate that alternative PCD pathways exist that may be
particularly relevant under pathological conditions. Indeed, a
variation of the classic form of apoptosis has been described;
it occurs in the absence of caspase activation, is associated
with less compact chromatin condensation (stage 1 chromatin
condensation), and has been defined as apoptosis-like PCD.
The best-characterized regulator of apoptosis-like PCD is
apoptosis-inducing factor, a mitochondrial flavoprotein with
oxidoreductase activity that translocates to the nucleus in response to defined cell-death stimuli (Cande et al. 2002).
Apoptosis-inducing factor, by itself, triggers partial nuclear
condensation and large-scale caspase-independent DNA fragmentation (Susin et al. 1999).
Necrosis, also referred to as accidental cell death, is characterized by rapid swelling of the dying cell, rupture of the
plasma membrane (as characterized by electron microscopy),
and release of the cytoplasmic content to the cell environment. Despite the profound effects of necrotic-like cell death
in pathological conditions, such as stroke, ischemia, and several neurodegenerative diseases (Stefani and Dobson 2003),
the molecular mechanisms underlying necrotic cell death are
poorly understood. Necrosis has traditionally been defined
as an unregulated (accidental) cell-death process that often
occurs under conditions of cellular injury (Barros et al. 2001a)
and is related to the loss of ion homeostasis (i.e., increase in
intracellular sodium concentration) and drastic decreases in
ATP levels (Majno and Joris 1995). However, in recent years,
an increasing number of reports indicate that cell death with
necrotic features occurs under normal physiological conditions and during development (Jaattela and Tschopp 2003;
Yuan et al. 2003). Also, evidence (Hetz et al. 2002b; Leyton
and Quest 2004) indicates that traditional apoptotic pathways may lead to necrotic cell death under certain conditions, and that specific mechanisms regulate this transition,
as will be discussed. In this context, it is important to mention that such forms of regulated necrotic cell death have
been defined as necrosis-like PCD, thereby emphasizing the
active nature of the process and distinguishing it from the
fast swelling and lysis normally observed in accidental
necrosis, which is a passive process. Such distinction is
essential because both forms of cell death are phenotypically
alike, but are distinct from apoptosis, and are probably caused
by different mechanisms (Jaattela and Tschopp 2003). In
particular, at late stages of both accidental necrosis and
necrosis-like PCD, nuclear morphology is similar and is
characterized by the absence of apoptotic features. However,
the rapid increase in cell volume observed before cell lysis
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can be used as a morphological manifestation of accidental
necrosis.
Cell death is often also associated with the presence of numerous cytoplasmic autophagic vacuoles of lysosomal origin.
Lysosomes have been referred to as suicide bags, because
they contain several unspecific digestive enzymes that, upon
release into the cytosol, cause autolysis and cell death (Bursch
2001). Autophagy, the process by which cells recycle cytoplasm and dispose of defective organelles (Shintani and
Klionsky 2004), also defined as type II PCD, plays a central
role in the maintenance of cellular homeostasis, and participates in the turnover of intracellular organelles and in the
regulation of proteins with a long half-life. Lysosome-mediated
cell death has been linked to the apoptotic pathway through
alterations in mitochondrial function (Boya et al. 2003a,
2003b). Cell death by autophagy may also lead to a necrotic
phenotype. However, identification of this form of PCD is
difficult, because death by autophagy does not display
easily identifyable characteristics, such as chromatin
condensation. The main criteria defining a cell-death
process, such as autophagy, is the appearance of doublemembrane-containing vacuoles in the cytosol, and fusion of
autophagosomes with the lysosomes (Bursch 2001). In addition, several genes relevant to autophagy, such as beclin 1
(an autophagic gene), are upregulated and used as indicators
of this process. More details concerning autophagy and the
molecular processes involved can be found in recent reviews
(Edinger and Thompson 2003; Shintani and Klionsky 2004).
General interest in understanding the mechanisms leading to
autophagy has increased considerably in recent years, because different anticancer drugs are believed to elicit their
cytotoxic effects by inducing autophagy (Gozuacik and
Kimchi 2004). Here, however, it is important to bear in mind
that under conditions of nutrient deprivation, autophagy is
thought to operate, at least initially, as a survival rather than
a suicide pathway (Klionsky and Emr 2000; Shintani and
Klionsky 2004).
In summary, the following criteria can be employed to
differentiate accidental necrosis from PCD-necrosis and autophagy. Accidental necrosis is characterized by rapid swelling
and a loss of plasma membrane integrity, frequently in connection with dramatic irreversible drops in ATP levels. In
contrast, PCD-like necrosis can be a relatively slow process,
observed as the consequence of chemical insult or downstream from death receptors, that also leads to membrane
rupture but is actively regulated at the molecular level.
Finally, autophagy is also a relatively slow process associated with the appearance of double-membrane vacuoles in
the cytoplasm, and extensive membrane-fusion events that
are controlled by the expression of autophagy-specific genes.
In the latter case, neither volume changes nor release of
cytosolic contents is observed, and the process can be associated with both cell survival and cell death.
As can be seen from this brief introduction, many excellent
reviews exist that summarize molecular events associated
with apoptosis or other forms of cell death. Here, we will
focus on the available literature that suggests that necrotic-like
cell death may not always be accidental, and that this form
of cell death may contribute to the regulation of cellular homeostasis under certain conditions.
© 2005 NRC Canada
Hetz et al.
Caspase-independent cell death and death
receptors
Two central pathways involved in the extrinsic stimulation
of apoptosis have now been linked to the activation of caspaseindependent (nonapoptotic) cell death. When caspases are inhibited, activation of the death receptors Fas (APO-1/CD95) and
tumor necrosis factor (TNF) receptor (TNFR)-1 can induce cell
death with necrotic-like features in certain cell types (Holler
et al. 2000; Vercammen et al. 1998a, 1998b). In general, activation of Fas and TNFR-1 involves the binding of their ligands (FasL or TNF-α, respectively) and the stabilization of
the trimeric form of the receptor, thereby allowing the assembly of the death-inducing signaling complex (Peter and
Krammer 2003). For example, activated Fas recruits the adaptor protein Fas-associated death-domain (DD) - containing
protein (FADD). The amino-terminal DD of FADD interacts
with a homologous DD within the prodomain of caspase-8
and (or) caspase-10, providing a platform for their activation. Activated caspase-8, in turn, activates downstream
effectors, such as the Bcl-2 family member Bid (Li et al.
1998) and caspase-3 (Nagata and Golstein 1995; Suda et al.
1993), ultimately leading to cell death. In addition, stimulation of Fas leads to receptor-interacting protein (RIP) 1
kinase recruitment, and activation of the transcription factor
NF-κB via interaction with TNFR-associated factors. Similar
protein complexes have been described for TNFR-1, which
first recruits TNFR-associated death-domain protein, leading
to RIP and caspase-8 activation (Peter and Krammer 2003).
Activation of the TNFR-1-pathway may, depending on the
cell type, lead to either apoptosis or necrosis-like PCD (Laster
et al. 1988). The mechanisms by which TNF triggers a
necrosis-like phenotype are poorly understood. Despite many
structural similarities between Fas and TNFR-1, FasL was
initially thought to trigger only apoptotic cell death. However, as will be discussed, inhibition of Fas-induced apoptosis
in L929 fibrosarcoma cells by a general caspase inhibitor
(zVAD-fmk) leads to cell death with necrosis-like characteristics that is mediated by oxygen radical production (Vercammen et al. 1998a, 1998b). Also, primary activated T cells
can be efficiently killed by FasL, TNF-α, and TNF-related
apoptosis-inducing ligand in the absence of active caspases
(Holler et al. 2000). These results suggest that Fas, like
TNFR-1, can trigger apoptotic or necrosis-like PCD.
In an early study, Vercammen et al. (1998a) showed that
murine L929 fibrosarcoma cells treated with TNF-α die rapidly by necrosis, owing to excessive formation of reactive
oxygen intermediates. In addition, overexpression of CrmA,
a serpin-like caspase inhibitor of viral origin, renders L929
cells 1000 times more sensitive to TNF-α-mediated cell
death. The presence of zVAD-fmk resulted in a rapid
increase of TNF-α-mediated production of oxygen radicals
that was associated with cell death. In addition, the same
group found that in L929 fibrosarcoma cells overexpressing
human Fas receptor, zVAD-fmk treatment augmented cell
sensitivity to Fas-mediated cell death with a necrosis-like
phenotype (Vercammen et al. 1998b). Interestingly, unlike
TNF-α, Fas-activation did not initiate NF-κB-dependent processes. These results demonstrated, for the first time, the
existence of 2 different pathways originating from the Fas
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receptor: a first leads rapidly to apoptosis, and, when this
apoptotic pathway is blocked by caspase inhibitors, a second
pathway, involving oxygen radical production, redirects the
cells to undergo necrosis-like PCD.
Additional evidence is available to support the idea that
alternative signaling pathways triggered by Fas can lead to
necrosis-like PCD. A caspase 8-deficient subline of human
Jurkat cells can be killed by the enforced oligomerization of
FADD under artificial conditions. Interestingly, the cell death
observed is not accompanied by caspase activation, and is
associated with some morphological changes typical of
necrosis (Kawahara et al. 1998). In addition, Matsumuraand
colleagues (2000) showed that the DD of FADD is responsible
for the FADD-mediated necrosis-like PCD pathway. This
process was accompanied by a loss of mitochondrial transmembrane potential, but not by the release of cytochrome c
from mitochondria. Thus, within the same cell, pathways
leading to apoptosis and necrosis-like cell death are initiated
by death receptors, but are differentially activated, depending on the nature of the stimuli. The results suggest that
caspase-dependent pathways leading to apoptosis are temporally faster and, when such rapid events are blocked, alternative pathways appear to result in necrosis-like cell death.
Interestingly, in L929 cells, both the TNF-α and FasL-induced
necrosis observed in the presence of the caspase inhibitor
zVAD-fmk are prevented by the serine protease inhibitor Ntosyl-L-phenylalanine chloromethylketone and the oxygen radical scavenger butylated hydroxyanisole, whereas Fas-induced
apoptosis is not affected, indicating that production of oxygen radicals is mediating necrosis-like PCD in this experimental system (Denecker et al. 2001). These data indicate
that cell-death mechanisms triggered by TNFR and Fas are
not identical, and that necrosis-like PCD may involve particular regulators not related to the canonical apoptotic pathway. Consistent with this idea, a recent study implicated
different FADD domains in the mediatiation of apoptosis or
necrosis-like PCD (Vanden Berghe et al. 2004).
Holler et al. (2000) reported that Fas triggering in
caspase-8-deficient Jurkat T lymphoma cells promotes cell
death associated with mitochondrial damage in the absence
of apoptotic markers. The authors showed that induction of
caspase-independent cell death was inhibited in primary T
cells deficient in either FADD or RIP. Similar observations
were made in models of TNF-α and TNF-related apoptosisinducing ligand toxicity (Holler et al. 2000). More important, RIP requires its own kinase activity for death signaling
and is independent of its known role in NF-kB activation,
suggesting that RIP may regulate, by phosphorylation, unknown factors involved in the activation of necrotic-like
PCD.
The complexity of caspase-independent cell-death regulation has increased in recent years, with the identification of
other molecular components associated with this pathway.
For example, inhibition of the chaperone Hsp90 with
geldanamycin blocked FasL-mediated necrosis-like PCD
(Lewis et al. 2000). As a possible mechanism, Hsp90 has
been found to regulate the turnover of RIP through the
proteasome pathway (Lewis et al. 2000). In addition, L929
cells undergoing necrosis-like cell death in response to
TNF-α were shifted to the apoptotic program in the presence
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of geldanamycin (Vanden Berghe et al. 2003). This shift was
blocked by CrmA but not Bcl-2 overexpression, suggesting
that the necrosis-to-apoptosis transition occurred through
activation of procaspase-8. A careful examination of the levels of different components of the TNFR-associated proteins
revealed that inhibition of Hsp90 alters the general protein composition of the TNFR-1 complex, probably favoring
caspase-8-dependent apoptosis (Vanden Berghe et al. 2003).
For example, geldanamycin treatment led to a proteasomedependent decrease in the levels of RIP, the inhibitor of
kappa B kinase-alpha, and, to a lesser extent, adaptors like
the NF-κB-essential modulator and TNFR-associated factor
2 (Vanden Berghe et al. 2003). The authors proposed that
the availability of particular proteins, such as RIP, FADD,
and caspase-8, may determine whether TNFR-1 activation
leads to apoptosis or to necrosis-like PCD. Similar results
were described by the same group when caspase-independent
cell death was initiated in L929 cells by the artificial
dimerization of FADD. Geldanamycin reverted the induction
of necrosis-like PCD to apoptosis in this model of FADDmediated cell death (Vanden Berghe et al. 2004).
Finally, using a genetic approach, in mouse embryonic
fibroblasts (MEFs), RIP, FADD, and TNFR-associated factor
2 (TRAF2) were shown to be critical components in the
sequence of events leading to TNF-α-induced necrosis-like
PCD upon caspase inhibition (Lin et al. 2004). Inhibitors of
NF-κB facilitated TNFR-induced necrosis-like cell death, but
other signaling events triggered by TNF-α (like JNK, p38,
and ERK activation) were not required for this process. Cell
death in this context depends on the generation of reactive
oxygen species, which was not observed in RIP(–/–),
TRAF2(–/–), or FADD(–/–) MEFs (Lin et al. 2004). These
data reinforce the notion that caspase-independent cell death
is an active regulated process, and that formation of signaling complexes at the cell surface is crucial in mediating
necrosis-like PCD via death receptors.
Caspase-dependent initiation of apoptosis
and necrosis by the Fas receptor
Our group also studied the signaling events involved in
FasL cytotoxicity in B and T lymphoma cells (Hetz et al.
2002b). As a first model for our studies, A20 B lymphoma
cells were used. Several known events linked to the onset of
apoptosis in A20 cells, including caspase-3 activation, cell
shrinkage, and DNA fragmentation, were all activated within
the first 3 h of Fas triggering. In these cells, the viability was
only partially restored in the presence of a caspase-3 inhibitor,
whereas the general caspase inhibitor zVAD-fmk completely
protected cells against FasL-induced death. Flow cytometric
analysis of cell-death markers was used to distinguish the 2
cell populations. In one, cell death was caspase-3-dependent
and paralleled by DNA fragmentation, cell shrinkage, and
nuclear fragmentation. In the other, cell death was caspase3-independent and occurred without any indication of DNA
fragmentation or nuclear condensation. In addition, these
cells increased in volume. Based on these criteria, the 2
propidium-iodide-positive populations of dead cells were
defined as apoptotic and necrotic cells, respectively, (see cell
and model in Fig. 1 and Hetz et al. 2002b). A key difference
here, with respect to previous reports, is that inhibition of
Biochem. Cell Biol. Vol. 83, 2005
caspases after Fas triggering was not necessary to induce the
alternative form of cell death. Interestingly, effects similar to
those observed in A20 cells were also detected in Jurkat T
cells, whereas for Raji B lymphoma cells lacking phosphatidylserine externalization and delayed ceramide production (Tepper et al. 2000), only Fas-induced apoptosis was
observed.
Interestingly, when caspase activity was analyzed in situ
in the A20 B lymphoma cells, caspase-3 activation was shown
to occur only in the shrunken-cell population (apoptosis),
whereas caspase-8 activation was detectable in both the apoptotic and necrotic populations (Hetz et al. 2002b). These
unexpected results identified, for the first time, that Fas
ligation leads simultaneously to apoptosis and necrosis-like
cell death, in which activation of initiator caspases, such as
caspase-8, represents a key initial step triggered by FasL
(Fig. 1).
As discussed above, initiation of dual Fas-signaling events
that lead to both apoptosis and necrosis-like PCD have been
described in several experimental systems when caspase
activation is blocked (Holler et al. 2000; Vercammen et al.
1998a). Such results suggest that necrosis is favored either
when pathways normally leading to apoptosis are blocked or
when an alternative caspase-independent pathway is triggered. Our results expanded on these observations by showing that apoptosis and necrosis-like cell death require
caspase activation to a different extent and can be triggered
in the absence of caspase inhibitors. Caspase inhibitors were
employed to help distinguish between the 2 types of cell
death. Low concentrations of zVAD-fmk (less than 1 µmol/L)
selectively reduced Fas-induced apoptosis without modulating necrosis, whereas at higher concentrations, both modes
of cell death were affected (Hetz et al. 2002b). Thus, elevated levels of initiator caspase-8 activity appear to favor
FasL-induced apoptosis and, as a prerequisite, caspase-3 activation. Under conditions in which stimulation via Fas leads
only to low levels of caspase-8 activity (i.e., presence of
zVAD-fmk, low levels of death receptor or ligand), apoptosis
may not be initiated and signaling events leading to necrosis
become the predominant cell death pathway. Based on these
findings, as a more general rule, it is possible that subthreshold stimuli may be employed to ensure cell death by
mechanisms other than apoptosis.
Role of ceramide, a lipid second
messenger, in Fas-mediated necrosis
In addition to caspase activation, treatment with either
FasL or TNF-α leads to the formation of ceramide (GarciaRuiz et al. 1997; Gudz et al. 1997). Sphingomyelin hydrolysis,
by either neutral (nSMase) or acidic sphingomyelinases
(aSMase), is generally implicated in ceramide production.
However, it has also been suggested that de novo biosynthesis
of ceramides plays a role in the induction of apoptosis (Hannun
and Obeid 2002; Ogretmen and Hannun 2004). In some experimental systems, apoptosis is accompanied by caspasedependent delayed (by several hours) ceramide production,
owing to the hydrolysis of sphingomyelin associated with a
loss of lipid asymmetry in the plasma membrane. As a result,
phosphatidylserine is externalized and sphingomyelin is internalized, acting as a substrate for cytosolic SMases, which
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Fig. 1. Possible signaling events in Fas-mediated cell death. The binding of FasL to Fas stabilizes the trimeric receptor and promotes
binding of the adaptor protein Fas-associated death-domain (DD) - containing protein (FADD) to the cytosolic domain of Fas. FADD recruits
procaspase-8 into the death-inducing signaling complex, thereby promoting its autoactivation. Downstream activation of the executioner
caspase-3 leads to cell death by apoptosis. Alternatively, caspase-8 also triggers temporally delayed generation of ceramides, possibly
by activating a neutral sphingomyelinase. Under certain conditions, ceramide signaling triggers cell death by necrosis-like programmed
cell death (PCD) or by autophagy. Induction of autophagy has been related to the activation of beclin 1 or BNIP3. Late production of
ceramides possibly triggers nonapoptotic cell death in those cells in which initial levels of caspase-8 activation is not sufficient to activate
caspase-3 and the apoptotic pathway. As another possibility, downstream of the Fas receptor, binding of receptor-interacting protein
(RIP) kinase activates a caspase-independent pathway and triggers necrosis-like PCD. This sequence of events involves reactive oxygen
species production and is negatively modulated by Hsp90 and the proteasome pathway that promote RIP degradation. Dotted lines
indicate tentative connections. PKB, protein kinase B; ROS, reactive oxygen species.
release ceramides. Interestingly, Raji B cells that are deficient in lipid scrambling do not produce ceramides upon Fas
activation; however, these cells still die by apoptosis upon
Fas triggering (Tepper et al. 2000).
Several reports have suggested that ceramide production
participates in the apoptotic cell death of lymphoid cells,
mainly by correlating the simultaneous appearance of apoptotic
markers with ceramide production (Garcia-Ruiz et al. 1997;
Gudz et al. 1997). However, experiments in which genetic
manipulation was employed to analyze the contribution of
different SMases in Fas-induced apoptosis failed to implicate any of these enzymes (Brenner et al. 1997; Cock et al.
1998; Tepper et al. 2001), arguing against a role for ceramide
in promoting apoptosis in such situations. Furthermore, experiments in HEK293 and HeLa cells established that ceramide
production is an initiator caspase-dependent proximal event in
Fas signaling that occurs independent of commitment to the
effector phase of apoptosis (Grullich et al. 2000).
As mentioned, and in contrast to the situation described
for A20 and Jurkat cells, Fas triggering in Raji B cells neither
leads to late ceramide production (Tepper et al. 2000) nor
results in Fas-induced necrosis-like PCD (Hetz et al. 2002b).
These observations indicate that lipid scrambling and ceramide
production may represent the crucial molecular events linking
the Fas receptor to necrosis. In our studies, lipid scrambling
was detectable in essentially all cells committed to death
(apoptotic and necrotic-like cells) after Fas triggering, and
was blocked with low concentrations of zVAD-fmk but
not with caspase-3 inhibitors, as described elsewhere (Tepper
et al. 2000). In agreement with such observations, caspaseindependent (Holler et al. 2000) or necrotic (Krysko et al.
2004) cell death is also associated with phosphatidylserine
externalization. Taken together, these observations link Fasinduced lipid scrambling, late ceramide production, and cell
death by necrosis. Furthermore, they suggest that caspase-3dependent apoptosis and other forms of cell death may not
be clearly distinguished when phosphatidylserine externalization is used as a criterion.
Ceramide production is specifically linked to the induction of necrosis, because cell-permeable C6- or C2-ceramide
induced cell death without caspase-3 activation, DNA fragmentation, cell shrinkage, or chromatin condensation (Hetz
et al. 2002b). Instead, cells increased in size and were filled
with vacuolar structures, resembling FasL-induced cell death
by necrosis-like PCD. Likewise, increases in endogenous
ceramide levels after treatment with bacterial SMase also
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promoted cell death with a necrotic phenotype. In addition,
the ceramide effect was shown to be dominant, in the sense
that the earlier ceramide was present, the higher the observed percentage of necrotic cell death. These data lead us
to speculate that after Fas triggering, ceramide production
downstream of caspase-8 may be temporally delayed, in
comparison to caspase-3 activation and apoptosis induction,
so necrosis is only triggered in those cells not already committed to apoptosis.
Ceramide signaling and cell death:
initiation of necrosis or autophagy?
Nonapoptotic cell death has been shown to be modulated
by the genetic manipulation of survival signaling pathways.
For example, when caspases are inhibited, cytotoxic agents,
such as staurosporine and dexamethasone, induce necrosis-like
cell death, which can be prevented by the overexpression
of Bcl-2 (Amarante-Mendes et al. 1998). Also, there are
several examples in which deletion of crucial apoptosisregulating genes in mice and Caenorhabditis elegans leads to
PCD by other mechanisms, most often necrosis-like PCD
(Yuan et al. 2003). For instance, Apaf-1-null embryonic stem
cells undergo nonapoptotic cell death after treatment with
various cytotoxic stimuli that can be inhibited by Bcl-2 overexpression (Haraguchi et al. 2000).
Other groups have also found that ceramide induces caspaseindependent cell death. Synthetic ceramides have been shown
to induce a necrosis-like morphology in neuroblastoma cells
(Ramos et al. 2003). Also, ceramides kill normal lymphocytes and cell lines in the absence of caspase activation, triggering a nonapoptotic morphology in dying cells (Mengubas
et al. 1999). In addition, ceramide-induced cell death in
leukemia cell lines was accompanied by minimal activation
of caspase-3, and was not inhibited by zVAD-fmk (BelaudRotureau et al. 1999). We confirmed a number of these observations in B and T lymphoma cells (Hetz et al. 2002b). In
addition, caspase-3-deficient cells have been shown to be
sensitive to ceramide cytotoxicity (Belaud-Rotureau et al.
1999). Remarkably, cell viability was restored when cells
were treated with the protease inhibitor leupeptin. Mochizuki
et al. (2002) reported that ceramide induced cell death in human glioma cells with a necrosis-like phenotype that was
efficiently inhibited by the activation of the AKT – protein kinase B pathway. This unexpected result reinforces the
emerging concept that signaling pathways controlling
apoptosis and necrosis-like PCD overlap. Taken together,
these observations suggest that nonapoptotic cell death triggered by ceramide is an active and regulated process.
Two recent reports have yielded new insights into the
mechanisms underlying ceramide-induced cell death. Using
different cell lines, ceramide was shown to stimulate autophagy
by 2 nonexclusive mechanisms (Scarlatti et al. 2004). In
human colon cancer HT-29 cells, C2-ceramide, a cell-permeable
ceramide analog, stimulates autophagy by increasing the intracellular pool of long-chain ceramides. Ceramides stimulated
the expression of the autophagic gene beclin 1 (Scarlatti et
al. 2004), linking, for the first time, known molecular mediators
of autophagy (Liang et al. 1999) to lipid second messenger
signaling. Ceramide also mediates the tamoxifen-dependent
accumulation of autophagic vacuoles observed in human
Biochem. Cell Biol. Vol. 83, 2005
breast cancer MCF-7 cells. Ceramide-dependent expression
of beclin 1 in tamoxifen-treated cells was impaired in the
presence of myrio, an inhibitor of the serine palmitoyltransferase, the rate-limiting enzyme of de novo synthesis of
ceramide. These data suggest a novel function for ceramide
in controlling a major lysosomal pathway, and provide a
molecular link between autophagy and cell responses to stress
(Scarlatti et al. 2004).
Another report linked ceramide toxicity to autophagy in
malignant glioma cells. Ceramide toxicity was accompanied
by several specific features characteristic of autophagy, such
as the presence of numerous autophagic vacuoles in the
cytoplasm, development of the acidic vesicular organelles,
autophagosome membrane association of microtubule-associated
protein light chain 3 (LC3), and a marked increase in expression of 2 forms of LC3 protein (LC3-I and LC3-II). They
also demonstrated that ceramide activates the expression of
death-inducing mitochondrial protein BNIP3 (Daido et al.
2004). BNIP3 is a new member of the Bcl-2 protein family;
it contains the Bcl-2 homology domain-3 (Hengartner 2000)
that has been shown to promote necrosis-like PCD when
overexpressed (Vande Velde et al. 2000). In this particular study,
the authors showed that BNIP3 triggered autophagy per se
via ceramide release (Daido et al. 2004).
In summary, taking ceramide as an example, signaling in
nonapoptotic PCD may be linked to common signaling components of the classic apoptotic pathway. In doing so, a
novel concept in cell-death regulation is emerging, in which
precisely coordinated sequences of molecular events, with at
least some elements in common, determine whether cells die
by apoptosis or by another form of PCD. These differences
are likely to have significant biological consequences at the
physiological and pathological levels, and to offer interesting
possibilities for therapeutic intervention once the governing
principles are understood.
Nonapoptotic PCD in physiology, pathology,
and disease
In contrast to apoptosis, cell death by a necrosis-like mechanism is typically associated with inflammation. This difference in the physiological response is related to the activation
or maturation of phagocytic cells, like macrophages and dendritic cells (Fadok et al. 2000; McDonald et al. 1999; Sauter
et al. 2000). Sauter et al. (2000) showed that immature dendritic cells phagocytose a variety of apoptotic and necrotic
cells. However, only exposure to necrotic cells provided the
signals required for dendritic cell maturation, resulting in the
upregulation of maturation-specific markers, costimulatory
molecules, and the capacity to induce antigen-specific CD4+
and CD8+ T-cells. Thus, dendritic cells are able to distinguish between the 2 types of dead cells and respond in a
distinct manner. The interaction between apoptotic and phagocytic cells induces an anti-inflammatory response (Fadok
et al. 2000), whereas necrosis appears to be critical for initiation of an immune response. Because phosphatidylserine
externalization is critical for the recognition of dead cells
(probably apoptotic and necrotic cells), the data discussed
reinforce the notion that lipid scrambling and phosphatidylserine externalization do not provide the molecular basis to
distinguish between cells dying by apoptosis and those
© 2005 NRC Canada
Hetz et al.
dying by necrosis, and, as a consequence, to elicit different
responses of the immune system.
Simultaneous or delayed activation of pathways leading to
apoptosis or necrotic-like PCD in the same cells is likely to
occur fairly frequently under pathological conditions (Ankarcrona 1998; Barros et al. 2001a; Dypbukt et al. 1994; Hetz
et al. 2002a; Jonas et al. 1994). One possibility is that catastrophic or accidental events rapidly deplete cells of ATP
and, in doing so, preclude organized cell demise by ATPrequiring mechanisms, such as apoptosis. Thus, necrosis occurs as a default pathway when apoptosis is blocked, as has
been suggested in models of accidental necrosis (Leist et al.
1997). Alternatively, the cell may actively undergo death by
activating pathways leading to necrosis. This could be
achieved by directly manipulating ATP levels and (or) other
mechanisms, including the production of reactive oxygen
species and the liberation of ceramides.
An interesting parallel can be drawn with models of
necrosis-like PCD in C. elegans. Mutations in a sodium
channel of the degenerin gene family of C. elegans that
increase channel activity cause neuronal degeneration through
necrotic events, including intracellular vacuole formation and
swelling. In mammals, the activity of sodium channels has
also been linked to cell death by accidental necrosis mediated by oxidative stress, a phenomena generally associated
with several pathological conditions (Barros et al. 2001a,
2001b). More recently, Simon et al. (2004) demonstrated
that hydroxyl radicals increase the open probability of a
roughly 20-pS calcium-activated, nonselective cation channel in liver cells, and reduce calcium dependence. Thus, the
presence of reactive oxygen species effectively modulates
the activity of these channels and, in doing so, may contribute to the regulation of necrosis.
Interestingly, loss-of-function mutations in calreticulin, a
major Ca2+-binding chaperone in the endoplasmic reticulum (ER), or double mutations in both the ryanodine and
inositol-1,4,5-trisphosphate receptors that block Ca2+-release
channels, suppress death in C. elegans by necrosis (Xu et al.
2001). Thus, an increase in cytoplasmic Ca2+ concentration,
elicited by release from the ER, may play a major role in
initiating necrosis-like cell death events triggered by abnormal functioning of plasma membrane Na+ channels. Conditions that activate accidental necrosis may be incompatible
with the execution of apoptotic or necrosis-like PCD pathways (such as fast swelling and lysis). The ability of high
levels of Ca2+ to induce swelling and disruption of mitochondria, causing permeability transition and loss-of-energy
metabolism, is likely a key event leading to necrosis. Interestingly, ceramide treatment has also been shown to increase
cytosolic and mitochondrial calcium levels (Belaud-Rotureau
et al. 1999; Pinton et al. 2001). Furthermore, ceramides have
been shown to directly modulate mitochondria function, for
instance, by inhibiting the mitochondrial respiratory complex III
(Garcia-Ruiz et al. 1997; Gudz et al. 1997; Quillet-Mary et
al. 1997). In this context, it is possible that the induction of
nonapoptotic cell death by ceramide may result from a
decrease in ATP levels due to mitochondrial dysfunction and
calcium release from the ER.
Alterations in ER homeostasis (denominated ER stress)
have been linked to specific apoptotic pathways associated
with alterations in calcium homeostasis and accumulation of
585
misfolded proteins in this organelle (Breckenridge et al. 2003).
Under pathological conditions that affect the nervous system,
ER stress plays a central role in controlling cell death observed
during stroke, ischemia, and neurodegeneration (Rao and
Bredesen 2004). For example, in models of Alzheimer’s disease (Nakagawa et al. 2000), prion disease (Castilla et al.
2004; Hetz et al. 2003), and Huntington’s disease (Nishitoh
et al. 2002), ER stress has been linked to neuronal dysfunction. Hence, under conditions of extensive ER stress, cells
may preferentially undergo necrosis-like cell death. It remains
to be investigated whether the common mediators of ER
stress that trigger apoptosis also participate in the induction
of nonapoptotic PCD.
Recent reports (Kegel et al. 2000; Petersen et al. 2001;
Stefanis et al. 2001) have shown that autophagic–necrotic
cell death is also involved in neurological diseases, highlighting the importance of understanding the mechanisms
associated with nonapoptotic cell death (Bahr and Bendiske
2002). Moreover, the relevance of such understanding is underscored by the fact that anticancer therapies are now known
to use nonapoptotic mechanisms to promote cell death and
tumorogenesis (Edinger and Thompson 2003; Liang et al.
1999; Paglin et al. 2001). This is relevant for the design of
novel therapeutic strategies, because cancer cells are often
resistant to apoptotic stimuli. In fact, animals with one of
the alleles for beclin 1 knocked out suffer from a high incidence of spontaneous tumors, suggesting that beclin 1 is a
tumor-suppressor gene (Yue et al. 2003). Examples that
emphasize such potential include a possible ceramide-based
therapy to treat gliomas (Daido et al. 2004), and the use of
anticancer drugs, such as tamoxifen and arsenic trioxide, to
induce tumor cell death by autophagy (Bursch 2001; Kanzawa et al. 2003). This may explain the fact that, in many
forms of cancer, increased resistance to apoptosis is a crucial
factor in cancer progression, and mutations in genes involved
in the regulation of apoptosis are genetically linked to some
forms of cancer. Thus, drugs that trigger nonapoptotic cell
death are likely to have therapeutic benefits. Along these
lines, a recent report indicates that, in knockout cells for crucial proapoptotic genes (such as Bax and Bak), stimulation
of cell death by classic proapoptotic stimuli promotes cell
death by autophagy as a default pathway (Shimizu et al.
2004).
Conclusions
Nonapoptotic cell death plays an important role in the
control of cell viability under normal physiological conditions, and when cell death is triggered by injury or disease.
Clearly, a more refined understanding of the molecular elements that determine whether cell death occurs by accidental
necrosis, necrotic-like PCD, autophagy, or apoptosis is required. The need for such insight becomes all the more
apparent when considering the fact that these different forms
of cell death share some characteristic features at the morphological level. Multiprotein complex formation is crucial
to signaling per se and to apoptosis in particular (Leyton and
Quest 2002, 2004). Recent advances indicate that peptidomimetics, which specifically target protein–protein interactions
relevant to either the extrinsic or intrinsic apoptotic pathway,
represent potentially valuable tools for selectively triggering
© 2005 NRC Canada
586
tumor-specific cell death (Li et al. 2004; Walensky et al.
2004). With this in mind, it is easy to appreciate how a
better understanding of nonapoptotic cell-death mechanisms
may translate into the development of more efficient therapeutic strategies for human disorders, such as cancer, autoimmune, and neurological diseases.
Acknowledgements
We thank Dr. Anna Schinzel and Dr. Scott Oakes for critical
readings of this article. Related ongoing research is supported by FONDAP 15010006, Wellcome Trust WT06491I/
Z/01/Z, ICGEB CRP/CH102–01 (to A.F.G.Q.), a CONICYT
student fellowship (V.T.), and a Post Doctoral Fellowship
from Damon Runyon Cancer Research Foundation (C.H.).
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