REVIEWS

Expansion and evolution of cell death

programmes
Alexei Degterev* and Junying Yuan‡
Abstract | Cell death has historically been subdivided into regulated and unregulated
mechanisms. Apoptosis, a form of regulated cell death, reflects a cell’s decision to die in
response to cues and is executed by intrinsic cellular machinery. Unregulated cell death
(often called necrosis) is caused by overwhelming stress that is incompatible with cell survival.
Emerging evidence, however, suggests that these two processes do not adequately explain
the various cell death mechanisms. Recent data point to the existence of multiple non-
apoptotic, regulated cell death mechanisms, some of which overlap or are mutually exclusive
with apoptosis. Here we examine how and why these different cell death programmes have
evolved, with an eye towards new cytoprotective therapeutic opportunities.

Cell death is a fundamental cellular response that has
a crucial role in shaping our bodies during develop-
ment and in regulating tissue homeostasis by elimi-
nating unwanted cells. The first form of regulated or
programmed cell death (PCD) to be characterized was
apoptosis, which was described in Caenorhabitis elegans
in the early 1990s1. Subsequent genetic analysis of mam-
malian apoptosis presented a more complex picture, in
which individual apoptosis genes from C. elegans have
expanded into large multi-protein families (FIG. 1). These
findings suggest that redundancy, functional special­
ization and compensatory regulation of mammalian
apoptotic signalling and execution might be important
features of mammalian apoptosis.
Because of its conserved and uniform nature, apop­
tosis is frequently defined mechanistically as a pathway
of regulated cell death that involves the sequential activa-
tion of caspases, a family of Cys proteases, and that is
controlled both positively and negatively by B-cell lym-
phoma protein-2 (BCL2) family members. Assays have
*Tufts University,
now been developed for multiple steps of the pathway,
School of Medicine, allowing the characterization of apoptotic death in vitro
Department of Biochemistry, and in vivo. Apoptotic cell death is characterized by
136 Harrison Ave., Boston, distinctive morphological features, including nuclear
Massachusetts 02111, USA.
fragmentation, membrane blebbing and the formation
‡
Harvard Medical School,
Department of Cell Biology, of apoptotic bodies (see Ref. 2 for further details), that
200 Longwood Ave., Boston, can be used to identify apoptotic cell death events.
Massachusetts 02115, USA. Genetic studies have shown that apoptosis has a sig-
Correspondence to J.Y. nificant role during normal mammalian development3,
e‑mail:
jyuan@hms.harvard.edu
especially in the central nervous system where genetic
doi:10.1038/nrm2393  deficiency of apoptotic genes (such as caspase‑9, apoptotic
Published online 16 April 2008 protease-activating factor-1 (APAF1), or BCL2-associated
X protein (BAX) and BCL2-antagonist/killer-1 (BAK)
double mutant mice) results in significant abnormalities4–6.
Apoptosis also functions to maintain homeostasis,
especially in the immune system, because it eliminates
unwanted cells. Dysregulation of apoptosis leads to vari-
ous human diseases, such as cancer and autoimmunity.
Inappropriate activation of cell death is also the leading
cause of tissue injury and functional decline in a large
number of acute diseases (such as stroke, myocardial
infarction and brain trauma) and chronic diseases (such
as diabetes and neurodegeneration). However, effective
cytoprotective therapies for these diseases remain a major
unmet medical need.
To a significant extent, the limited success of cyto­
protective drug development can be traced to the simpli-
fied view that cell death is either intrinsically regulated
by apoptosis or that it is unregulated, caused by over-
whelming stress (so-called necrosis). Necrosis possesses
characteristic features, such as organelle swelling, mito-
chondrial dysfunction, massive oxidative stress and rapid
plasma-membrane permeabilization, that are thought to
be indicative of the catastrophic nature of cell death, rather
than a result of cellular regulation. The general view of
the relationship between apoptosis and necrosis is that
milder insults to the cell cause apoptosis, whereas more
intense insults induce uncontrollable necrosis7. It is thought
that such an apparently unregulated — and hence untarg-
etable — process accounts for the bulk of cell death events in
acute pathologies. However, in the past few years, evidence
has emerged for a number of regulated non-apoptotic
cell death pathways, including some with morphological
features that were previously attributed to necrosis8.

378 | May 2008 | volume 9 www.nature.com/reviews/molcellbio

© 2008 Nature Publishing Group

It has become clear that the simple apoptosis–necrosis
classification does not adequately represent the complex-
ity of endogenous cell death regulation. We are now just
beginning to appreciate the important functions of reg­u­
lated non-apoptotic cell death in homeostatic regulation
and development of human disease.
In this review, we first provide a brief overview of
apoptosis and then examine how mammals might have
acquired more apoptotic genes and more complex
apoptotic regulation through gene duplication. We then
describe examples of regulated non-apoptotic cell death
pathways and consider the possible relationships and
evolutionary origins of the diverse cell death processes.
Apoptosis — the fundamental mechanism of PCD
The mechanism of PCD elucidated by genetic studies in
the nematode C. elegans defines a primordial paradigm of
apoptosis9. In C. elegans, the activation of PCD is controlled
by an elegant and simple pathway (FIG. 1). The initiation
of apoptosis is regulated by transcriptional upregulation of
egl‑1, a pro-apoptotic BCL2 homology-3 (BH3)-only member
of the BCL2 protein family10. Binding of EGL‑1 to anti-
apoptotic CED‑9 relieves the inhibition that CED-9 exerts
on the adaptor protein CED‑4, allowing CED‑4 to bind
and activate the Cys protease CED‑3, which in turn cleaves
multiple specific cellular substrates to execute cell death11.
Analysis of apoptosis in mammalian cells has led to
the identification of multiple mammalian homologues
for each class of the C. elegans CED proteins. Extensive
studies have revealed that the mechanism of mam­malian
apoptosis is similar to that of C. elegans, although it
has become much more complex (FIG. 1). For example,
although transcriptional upregulation of pro-apoptotic
members of the BCL2 family can still play a part in the
initiation of apoptosis in mammalian cells12, many BH3-
only, pro-apoptotic BCL2 family members can be activ­
ated in a number of different ways (including cleavage,
phosphoryl­ation, myristoylation and ubiquitylation13–16)
to regulate the initiation of apoptosis17.
In mammalian cells, the activated EGL‑1-like BH3-
only members of the BCL2 family also inhibit CED‑9-like
anti-apoptotic members of the BCL2 family (such as BCL2,
BCL-XL and MCL1) by direct interaction17 (BOX 1; FIG. 1).
However, a major mechanism by which anti-apoptotic
members of the BCL2 family protect cells is not through
the direct inhibition of caspase activation at the level of
adaptor molecules (for example, the mammalian CED‑4-
like APAF1 protein), but by protecting the integrity of
the mitochondria (BOX 1; FIG. 1). BH3-only factors bind
and antagonize anti-apoptotic BCL2 family members
that reside in the outer mitochondrial membrane. Anti-
apoptotic BCL2 proteins exert their activity by preventing
the pro-apoptotic multidomain BCL2 family members
BAX and BAK from causing mitochondrial damage18,
and binding of BH3-only proteins relieves this inhibi-
tion. Additionally, a subclass of BH3-only factors might
directly induce the formation of a BAX–BAK channel18,
BH3-only member although how much this direct mechanism contributes
Pro-apoptotic BCL2 family
member possessing only a
to apoptosis remains a subject of debate19. BAX and
BCL2 homology-3 (BH3) BAK have been proposed to form an oligomeric (at least
domain. tetrameric or larger6) channel that leads to mitochondrial
nature reviews | molecular cell biology volume 9 | May 2008 | 379
© 2008 Nature Publishing Group
REVIEWS
C. elegans Mammalian
Upstream signal Upstream signal
(transcription) (processing, transcription)
Activated
BH3-only
EGL-1 proteins
Mitochondria
CED-9 BCL2
BAX/BAK
Cytochrome c
release
APAF1
CED-4
Apoptosome
Caspase-9
Caspase-3
CED-3
Cell death Cell death
Figure 1 | Evolutionary expansion of C. elegans
apoptotic machinery in mammalian
Nature cells. Side-by-side
Reviews | Molecular Cell Biology
comparison of the Caenorhabditis elegans CED protein
pathway and the core apoptotic machinery in mammalian
cells shows the conservation of the general outline of the
pathway. Extension of the apoptotic machinery can also be
observed at every step of the pathway, including multiple
B-cell lymphoma protein-2 (BCL2) homology-3 (BH3)-only-
protein activating signals, complex regulation of the BCL2
family and the addition of mitochondrial cytochrome c
release, which drives the formation of an apoptosome and
activation of the upstream caspases (first caspase‑9 and
then the executioner caspases, such as caspase‑3 and
caspase‑7). Added complexity is provided by the existence
of multiple family members in each class of the apoptotic
regulators, with both redundant and non-redundant
functions. These regulators provide ‘fail-safe’ apoptosis
machinery that can generate specialized responses to
various upstream stimuli. Possible direct activation of
BAX and BAK by BH3-only proteins is indicated by a dotted
line. APAF1, apoptotic protease-activating factor-1; BAK,
BCL2-antagonist/killer-1; BAX, BCL2-associated X protein.
damage and cytochrome c release. In some cases, BAX and
BAK can act through interactions with the components
of the mito­chondrial permeability pore, namely with the
voltage-dependent anionic channel (VDAC)20. Mitochon­
drial damage might also be caused by BAX- and BAK-
independent mechanisms, such as those induced by intra­
mitochondrial K+ influx, or caused by the direct action
of caspase‑2 on mitochondria (which occurs, curiously,
independently of the protease activity of caspase-2)20.
Mitochondrial damage and the release of mitochon-
drial proteins amplifies apoptotic signalling in mam­
malian cells, a step that is considered to be less important
for apoptosis in lower organisms, including flies and
REVIEWS

Box 1 | Major classes of apoptosis mediators

Caspases Caspases
Caspases are a family of Cys proteases (humans have Prodomain p20 p10
11 caspases) that cleave their substrates after Asp residues. Executioner caspases
Caspases contain three main domains: a prodomain and
large (p20) and small (p10) catalytic subunits. The large Activator caspases
domain contains the active site Cys residue. Activation of DED, CARD
caspases involves the proteolytic cleavage of zymogens, the Inflammatory caspases
removal of the prodomain and separation of the p20 and DED, CARD
p10 subunits, or allosteric conformational changes. The
prodomains of activator and inflammatory caspases contain BCL2 protein family
protein–protein-interaction domains (such as the caspase- BH4 BH3 BH1 BH2 TM
recruitment domain (CARD) and the death-effector domain Anti-apoptotic
(DED)) that link them to apoptosis signalling molecules21.
Multidomain pro-apoptotic
BCL2 family
The B-cell lymphoma protein-2 (BCL2) family is subdivided BH3-only
into three sub-classes: anti-apoptotic (such as BCL2,
BCL-XL and MCL1), multidomain pro-apoptotic (BAX and
BAK) and BH3-only (such as BID, BIM, BAD, NOXA and APAF1, NLR and PIDD adaptors
PUMA)17. The BH1, BH2 and BH3 domains of multidomain PYD, CARD NACHT NAD LRR
family members form a hydrophobic cleft that serves as a NLRs
heterodimerization interface for the BH3 domains of
BH3-only proteins. The BH4 domain of anti-apoptotic CARD NB-ARC WD-40
BCL2 family members directly interacts with a voltage- APAF1
dependent anion channel and inhibits apoptotic LRR ZU-5 ZU-5 DD
mitochondrial changes113. Anti-apoptotic BCL2 family PIDD
members can be cleaved by caspases, which results in the
loss of the BH4 domain and in the proteins exhibiting pro-apoptotic, rather than anti-apoptotic, activity114.
BH1/BH2/BH3, BCL2 homology‑1/2/3 domain; TM, transmembrane domain. Nature Reviews | Molecular Cell Biology

APAF1, NLR and PIDD adaptors

NLR (nucleotide-binding and oligomerization domain (NOD)-like receptor), APAF1 (apoptotic protease-activating factor-1)
and PIDD (p53-induced protein with a death domain) activate caspases following pathogenic infection (caspase‑1),
cytochrome c release (caspase‑9) and genotoxic stress (caspase‑2), respectively. The NLR family (22 proteins in humans) can
be subdivided into NOD factors (which sense bacterial peptidoglycans and signal through RIP2 to activate NF‑κB),
inflammasome-forming NALPs, IPAF, NAIP and CIITA (which regulates major histocompatibility (MHC) class II transcription).
Activation of APAF1, NLRs and PIDD proteins leads to the formation of large multimeric caspase-activating complexes115,116.
Caspase recruitment can be direct (through CARD domains) or can involve additional adaptors (such as RAIDD (also known
as CRADD) interacting with the death-domain (DD) of PIDD116). The nucleotide-binding and oligomerization domain (the
NACHT domain) and NB‑ARC domains are required for oligomerization. The NB‑ARC domain of APAF1 also binds to dATP,
which is required for apoptosome formation. The WD‑40 repeats of APAF1 bind cytochrome c. The Leu-rich repeats (LRRs)
of NLRs probably have a key role in sensing pathogen-associated molecular patterns83. CIITA, major histocompatibility
complex (MHC) class II trans-activator; IPAF, ICE-protease activating factor; NAIP, neuronal apoptosis-inhibitory protein;
NALP, NACHT‑, LRR- and pyrin domain (PYD)-containing proteins; NF-κB, nuclear factor-κB.

worms. Cytochrome c, which is released from damaged
mitochondria, promotes the formation of a heptameric
‘apoptosome’ megacomplex of APAF1 and caspase‑9 (a
member of the CED‑3-like Cys protease family). This
leads to the conformational change and activation of
caspase‑9 (FIG. 1). Activated caspase‑9 in turn cleaves
and activates downstream caspases, including caspase‑3,
caspase‑6 and caspase‑7, that carry out the execution
phase of apoptosis21.
In addition to the intrinsic apoptosis pathway, which
resembles PCD in C. elegans, mammalian cells also pos-
sess an extrinsic apoptosis pathway, which is induced by
pro-apoptotic and pro-inflammatory cytokines (such as
FAS ligand (FASL) and tumour-necrosis factor‑α (TNFα),
which are ligands for the death-receptor family)22. By bind-
ing to death-domain receptors, FASL and TNFα induce
the formation of specific intracellular death-induced
signalling complexes (DISCs23), which activate upstream
caspases such as caspase‑8. The activation of caspase‑8 can
in turn cleave downstream caspases, such as caspase‑3 and
caspase‑7, to execute cell death; alternatively, caspase‑8
can cleave the BH3-only pro-apoptotic protein BID,
which in turn amplifies the cell death signal by causing
mitochondrial damage and cell death24. The development
of cytokine-mediated apoptosis programmes in higher
multicellular organisms provides a crucial way to coordin­
ate the regulation of cell numbers at the organismal
level in response to the environmental stimuli.
Evolutionary expansion of apoptosis
Although the core regulators of PCD in C. elegans consist
of only 4 genes — egl‑1, ced‑3, ced‑4 and ced‑9 — each
has multiple mammalian homologues. On the basis of
close homology in the key regions, the mammalian ced-
like genes probably arose through gene duplication and
might have evolved and been selected during evolution
to meet the challenges that highly complex multicellular
organisms face.

380 | May 2008 | volume 9 www.nature.com/reviews/molcellbio

© 2008 Nature Publishing Group

Anoikis
Cell death caused by cell
detachment from the matrix.
Linker cell
A cell in a C.elegans embryo
that is positioned between the
gonad and the cloacal tube.
Type I cell death
A term introduced as part of a
morphological classification of
developmental cell death.
Type I death shows the
morphological features of
apoptosis.
nature reviews | molecular cell biology volume 9 | May 2008 | 381
Multiple genes enable functional specialization. One
direct consequence of this gene duplication is specifica-
tion — different apoptosis regulators respond to differ­ent
pro-apoptotic signals. The mammalian caspase family
provides an excellent example of specification. Different
caspase family members possess distinct functions in
mammalian cells that are predominantly based on their
subcellular localization and protein–protein interactions
rather than on their substrate specificities25. Mammalian
caspases were initially assigned to three major classes:
apical or activator caspases, such as caspase-2, -4, ‑8, -9,
‑10 and ‑12, which initiate the caspase cascade in apop-
tosis; executioner caspases, such as caspase‑3, ‑6 and ‑7,
which act in the downstream execution steps of the proc-
ess; and inflammatory caspases, such as caspase‑1, -5
and ‑11, which mediate cell death and inflammatory
responses. Further analyses suggest that there might be
additional important distinctions between family mem-
bers in the same class. Activator caspases have distinct
roles that depend on the activating complexes that they
are recruited to. For example, caspase‑8 specifically
contributes to death-receptor signalling26 and normal
pro­liferation of lymphocytes27; caspase‑2 mediates
genotoxic stress-mediated death28; mouse caspase‑12
and human caspase‑4 mediate endoplasmic reticulum
(ER)‑stress-mediated death29,30; and caspase‑9 is activ­
ated by the apoptosome downstream of cytochrome c
release31. Such division of labour might provide a sensi-
tive mechanism to allow complex multicellular organ-
isms to detect and differentially respond to distinct
environmental stimuli.
A similar division of labour has been observed for
the members of the BCL2 family. BCL2 family members
can be subdivided into three major classes on the basis of
structural and functional differences17 (BOX 1). However,
recent studies suggest that subtle but important differ-
ences exist in each sub-class. In particular, although all
BH3-only factors possess a similar EGL‑1-like mode
of action, they can be activated non-redundantly by
particular types of apoptotic signals. For example, BID
mediates signalling by the TNF-family and by genotoxic
stress16,32; BIM plays a key part in inducing death in lym-
phoid and myeloid cells following cytokine withdrawal33;
NOXA and PUMA signal downstream of p53 (Ref. 34);
and BMF has a key role in anoikis35. The specificities of
BH3-only factors not only result from their interactions
with upstream regulators, they also result from their
distinct binding preferences for different multidomain
BCL2 family members. For example, the BH3 domain of
BAD binds BCL2 and BCL-XL, whereas the BH3 domain
of NOXA displays selectivity towards MCL1 and the
BCL2-related protein A1 (Ref. 36). Such selectivity in
the inter­actions of different members of the BCL2 family
provides an important mechanistic basis for activating
distinct apoptosis signalling pathways in response to
different pro-apoptotic stimuli.
The benefits of redundancy. An additional and non-
conflicting significance of the multiplication of apoptosis
regulators is the benefit that is provided by redundancy.
The redundancy of apoptosis programmes in mammalian
© 2008 Nature Publishing Group
REVIEWS
cells is shown by the compensatory upregulation
of caspases in different caspase mutant mice — the loss of
one caspase can be compensated by the upregulation
of another caspase37,38. Although the specification of
different apoptotic regulators in a signal and/or subcellu-
lar compartmentalization manner provides a mechanism
to fine-tune cellular responses, danger might arise if a
specified response is lost because of a genetic mutation.
In this case, it appears that upregulation of an alternative
caspase, or of its regulators, can compensate for the loss
of a specific caspase.
Although different caspases exhibit specificity towards
certain cleavage sites39, such specificity is relative. When
present in a sufficient concentration and given sufficient
incubation time, most caspases can cleave most, if not
all, of the caspase substrates that have been identified to
date. The preferential cleavage of a selective subgroup
of caspase substrates in the early phase of apoptosis
may only reflect the proximity of the substrates to the
activ­ated caspases at that specific time or they may serve
to modulate the kinetics of the process. The ability of
multiple caspases to cleave common substrates might
serve to insure the execution of apoptosis even when
one caspase is lost. Furthermore, although upstream
caspases can be activated preferentially during the early
stages of apoptosis (such as caspase-8 and ‑10, which are
preferentially activated in response to a FASL or TNFα
signal26), all caspases are cleaved and activated during
the later stages of apoptosis. Therefore, all of the caspases
may contribute to the execution of apoptosis21. Apoptosis
in mammalian cells is genetically programmed to maxi-
mize the ability to kill the cells once an appropriate order
is received.
Expansion or reduction. Although the findings dis-
cussed above lead us to suggest that apoptotic pathways
expanded during evolution, another view is also poss­
ible. C. elegans, along with its evolutionary relatives,
might have experienced a reduction in the complexity
of the apoptotic machinery from primordial ancestors
with apoptotic machinery that might have been closer
to that of humans. However, we find this scenario less
likely, because no such developed apoptotic machinery,
resembling that of mammals, has been identified in more
primitive organisms, such as plants, fungi and bacteria.
Non-apoptotic mechanisms of cell death
Emerging evidence suggests that apoptosis is not the
only mechanism of cellular suicide, but rather that cells
might choose one of many mechanisms to die when
they are ready, with apoptosis often representing the
top choice. In C. elegans, most developmental cell death
events occur through the apoptotic mechanism and only
rare events, such as the developmental death of a linker
cell, are non-apoptotic40. Type I cell death, which displays
the characteristic morphology of apoptosis as discussed
above, is also the most frequently observed form of
death during normal mouse development2. Although
induction of morphologically non-apoptotic develop-
mental cell death becomes prominent in mice in vivo
when apoptotic machinery is genetically disrupted
REVIEWS

Box 2 | Three pathways of non-apoptotic cell death

a Autophagic cell death b Necroptosis c PARP1-mediated cell death
TNFα DNA DNA
damage degradation Cell
death
TNFR
Cell PARP1 Nucleus
LC3-II membrane
Death RIP1 NAD+ PAR
domain Specific loss polymer
ATG5–ATG12, Death
initiation
ATG16L BCL2, domain
BCL-XL TRAF2
Beclin-1, UVRAG Kinase RIP1
VPS34, Energy
VPS15 Lipoxy- Acid collapse
genases SMase Ceramide JNK
WIPI ULK mTOR
Autophagy Rac1/NADPH JNK
ATG9 PAS oxidase AIF
Mitochondrial Mitochondrial Mitochondrial
dysfunction ROS dysfunction
Autophagosome formation Regulated necrosis Cell death Mitochondria
Autophagic cell death. Eighteen yeast autophagy-related (ATG) genes, which are required for autophagosome (AP)
formation, have been identified (AP–Atg proteins)117. Several mammalian homologues have been identified (shown in
figure panel a). The activity of the class III phosphatidylinositol 3-kinase complex is subject to inhibition by B-cell
Nature Reviews | Molecular Cell Biology
lymphoma protein-2 (BCL2) and to activation by the tumour suppressor UVRAG (UV radiation resistance-associated
gene protein). Nutrient-deprivation signalling induces autophagy through the suppression of mammalian target of
rapamycin (mTOR) activity. Functional analysis has implicated the same core ATG proteins in the formation of different
types of autophagosomes, including those that have pro-death roles118. The molecular machinery of mammalian
autophagosomes is still only partially characterized. Intrinsic differences in the composition of autophagosome protein
machinery might determine its function as a pro-survival mechanism, rather than a pro-death mechanism. PAS,
phagophore assembly site. See REF.117 for further details. This panel is modified, with permission, from ref. 117
(2007) Cold Spring Harbor Laboratory Press.
Necroptosis. The activation of RIP1 kinase increases reactive oxygen species (ROS) production (from the mitochondrial
respiratory chain and the RIP1–Rac1–NADPH oxidase complex) and activates c-Jun N-terminal kinase (JNK) kinase
(which might be crucial for the execution of necroptotic cell death in some cell types)119,120. Autophagy can be prominently
activated during necroptosis, but it only appears to contribute to cell death in some cell types63. Other execution steps,
including the activation of phospholipase A2, lipoxygenases121 and acid sphingomyelinase122, have been described.
The exact roles of these steps remain to be elucidated. Given the similarity of many of the downstream execution steps
in necroptosis to those attributed to ‘classic’ unregulated necrosis, the main difference between these two mechanisms
might be in the method of activation (regulated by internal signalling mechanisms rather than caused by overwhelming
stress) of similar relatively nonspecific execution events. SMase, sphingomyelinase; TNFα, tumour-necrosis factor-α;
TNFR, TNFα receptor.
PARP1-mediated cell death. Two pathways are shown: energy collapse and apoptosis-inducing factor (AIF) translocation.
In addition to the cell death role, poly(ADP–ribose) polymerase‑1 (PARP1) is involved in initiating DNA repair. Genetic
ROS deletion of PARP1 leads to sensitivity to DNA-damaging agents123. Oxidoreductase activity of AIF also has a homeostatic
(Reactive oxygen species). role, as it is crucial for mitochondrial complex I function124 and a mutation in mice that is associated with severe
Highly reactive intermediates progressive neurodegeneration has been mapped to AIF124. TRAF2, TNF receptor-associated factor-2.
in the reduction of oxygen to
water. These are produced, for
example, as a byproduct of
oxidative phosporylation in the (such as in caspase-deficient motor neurons41), one Type II cell death. Type II cell death is characterized
mitochondria. might question whether non-apoptotic cell death can by the accumulation of double-membrane-enclosed
also occur under normal conditions when apoptosis is vesicles. These vesicles are characteristic of auto­phagy
Type II cell death
A term introduced as part of a
possible. One could argue that non-apoptotic mecha- and so type II cell death is often called autophagic cell
morphological classification of nisms represent rudimentary back-up forms of cell death death, although the role of autophagy in this type of
developmental cell death. that are only relevant in rare circumstances when the cell death is still under debate2. Autophagy is an evolu-
Type II death shows the apoptotic machinery is genetically unavailable. However, tionarily-conserved intracellular catabolic mechanism
morphological features of
an understanding of the molecular mechanisms under- that operates at low levels under normal conditions to
autophagy.
lying non-apoptotic cell death in vitro has recently begun mediate the degradation of cytoplasmic components,
Necroptosis to emerge, and we are now beginning to appreciate the protein aggregates and expired intracellular organelles
A process of regulated non- importance of these processes. Here, we summarize (for example, the ER and mitochondria) by forming
apoptotic cell death displaying the data that describes three emerging regulated non- double-membrane-enclosed vesicles called autophago-
necrotic morphology, which
can be induced by death
apoptotic cell death pathways: type II cell death, necroptosis somes. The contents of autophagosomes are degraded
domain receptors through RIP1 and poly(ADP–ribose) polymerase‑1 (PARP1)-mediated by lysosomal enzymes after autophagosomes fuse with
kinase activity. necrotic death. lysosomes.

382 | May 2008 | volume 9 www.nature.com/reviews/molcellbio

© 2008 Nature Publishing Group

CD4+ T cell
One of a subpopulation of
mature T cells that express the
CD4 receptor.
NF-κB
(Nuclear factor-κB). A family of
transcription factors that are
activated by many
inflammatory signals.
Cyclophilin D
A mitochondrial matrix protein
that is associated with the
mitochondria permeability
pore.
nature reviews | molecular cell biology volume 9 | May 2008 | 383
Under normal conditions, autophagy has an important
role in maintaining intracellular homeostasis. In this role,
autophagy removes damaged or dysfunctional organelles
and misfolded proteins that can be detrimental to sur-
vival (for example, of neurons)42. Under conditions of
nutrient deprivation, autophagy promotes cell survival
by degrading disposable intracellular contents, thereby
generating energy and building blocks for protein syn-
thesis. Autophagy is regulated by a large group of ATG
(autophagy-related) genes that are conserved from yeast
to humans (BOX 2).
Activation of autophagy during Drosophila melano­
gaster metamorphosis has been well established43. Using
genetic elimination of Atg genes, it was recently shown
that a lack of autophagy attenuated the degradation of
D. melanogaster salivary glands by blocking PCD44. Thus,
this study provides evidence for the role of autophagy in
developmental cell death. However, the lack of cell death
defects in autophagy-deficient mutant mice (such as ATG5
or ATG7 mouse mutants), as well as in ATG7-deficient
Drosophila, raises the question of whether autophagy
per se or only part of the autophagy pathway is involved
in type II cell death during development2,45–48.
Autophagy might contribute to cell death that is
induced by viruses. Human immunodeficiency virus‑1
(HIV1) induces the accumulation of Beclin-1 protein and
cell death in uninfected bystander CD4+ T cells when the
HIV1 envelope protein (Env) interacts with the chemo­
kine receptor CXCR4 (Refs 49,50). Although the mecha-
nism by which autophagy is activated by Env is not yet
clear, this result suggests that autophagy might contribute
to cell death in a non-cell-autonomous manner, providing
a mechanism by which a virus might induce cell death
independently of viral replication.
Autophagy can assume the killer role when apoptosis is
unavailable51. For example, autophagy mediates cell death
in apoptosis-deficient BAX–/– BAK–/– cells in response
to genotoxic or ER stress stimuli52,53. It is possible that
autophagy is induced, albeit at low levels, under normal
conditions, but that it becomes exacerbated in response to
cellular stress during apoptosis-deficient conditions to pro-
mote cell death. In this regard, it is interesting to note that
Beclin-1 — the mammalian homologue of yeast Atg6 and
a regulator of the type III phosphatidylinositol 3-kinase
VPS34 — has a BH3 domain54 and interacts with BCL2,
which reflects the convergent regulation of apoptotic and
autophagic cell death55. Furthermore, a subset of BH3-only
pro-apoptotic BCL2 family members, including BNIP3
and BIK, can induce the activation of autophagic cell
death56,57. Although the mechanism by which BNIP3 and
BIK induce autophagy is not clear, and although it remains
to be seen whether autophagy is induced as a secondary
consequence of mitochondrial damage, such studies
raise the possibility that autophagic cell death might be
induced in a manner similar to that of apoptosis.
Necroptosis as a form of regulated necrosis. Necroptosis
(BOX 2), which represents a type of programmed necro-
sis, is another intriguing example of a regulated non-
apoptotic cell death mechanism. Its discovery was prompted
by observations that classic apoptotic stimuli, such
© 2008 Nature Publishing Group
REVIEWS
as death-domain-receptor engagement by correspond-
ing ligands, can lead to non-apoptotic cell death (as
assessed using morphological criteria) when apoptosis
is inhibited by caspase inhibitors58–61 or through muta-
tions in caspase‑8 or FAS-associated death-domain
protein (FADD)62–65. Although necroptosis is activated
by the same stimuli that initiate apoptosis, the morpho­
logical features of necroptosis — organelle swelling,
rapid mitochondrial dysfunction, plasma membrane
permeabilization and lack of nuclear fragmentation
— are characteristic of pathological necrosis, which
is presumed to be unregulated death that is caused by
overwhelming stress8,66.
Emerging evidence suggests that the initiation of the
necroptotic programme by TNFα occurs at the receptor
level through the recruitment and activation of an intra­
cellular signalling complex that involves the adaptor mole­
cule RIP1 (but not the TRADD–RIP1 complex, which
mediates the activation of nuclear factor-κB (NF‑κB)
and apoptosis)67. Activation of necroptosis requires the
kinase activity of RIP1, which is not required for NF‑κB
and apoptosis signalling64. The distinct nature of apop-
tosis and necroptosis was further emphasized by the
discovery, in a random cell-based screen, of a series of
structurally distinct small molecule necrostatins, all
of which specifically and efficiently inhibit necroptosis,
but not TNFα-induced apoptosis63,68–70. The mechanism
that leads to the execution of necroptosis downstream
of RIP1 kinase activation remains unclear. RIP1 is
translocated into the mitochondria, which leads to the
disruption of the association of ADP–ATP translocase
(ANT) with cyclophilin D (Ref. 71) and this might explain
the rapid mitochondrial dysfunction that is associated
with necroptosis63. Interestingly, the protective effect of
the necroptosis inhibitor necrostatin‑1 in an in vivo heart
ischaemia–reperfusion injury was dependent on the expres-
sion of cyclophilin D (Ref. 72). However, the mechanistic
details of this step remain to be determined.
PARP1-mediated necrotic death. PARP1 is a nuclear
enzyme that has a key role in maintaining genome
stability. PARP1 is rapidly activated by DNA-strand
breaks and recruits DNA-repair factors by attaching
ADP–ribose units to chromatin-associated proteins.
Loss of PARP1 leads to an increased sensitivity to DNA
damage73, which prompted the development of PARP1
inhibitors as chemopotentiators of DNA-damaging anti-
cancer agents74. However, over-activation of PARP1 can
lead to caspase-independent cell death75.
PARP1 can mediate cell death in a number of differ-
ent scenarios (BOX 2). In one scenario, alkylating DNA
damage promotes rapid PARP1-mediated depletion
of cytosolic NAD+, which leads to necrotic death by
‘energy collapse’ in glycolytic cells76 (these cells depend
on cytosolic NAD+ for glycolysis and energy genera-
tion). This mechanism can be viewed as an extension
of the genome-surveillance function of PARP1, as
it provides an elegant way to differentially regulate
DNA-damage responses in rapidly proliferating glyco­
lytic cells and in cells in a vegetative state, relying on
mitochondrial respiration for maintaining ATP levels76.
REVIEWS
Box 3 | The contribution of non-apoptotic cell death to pathological injury
Recent studies have shown that non-apoptotic cell death is not just a nuisance of
in vitro experimentation — it makes important contributions to pathological
regulation in vivo.
Autophagy has been prominently observed in various disease models and its
inhibition can provide therapeutic benefit. For example, recent studies suggest that
Beclin-1-dependent autophagy promotes injury in mouse models of heart ischaemia–
reperfusion injury and heart failure125,126. In addition, autophagy suppresses apoptosis
in MYC-dependent lymphomas, promoting tumour growth127. Conversely, genetic
analysis has clearly established that Beclin-1 functions as a tumour suppressor128.
Autophagy promotes the survival of cancer cells that express apoptosis-inhibiting
BCL2 family members under the conditions of hypoxia129. However, inhibiting
autophagy under such conditions unleashes necrosis, which might promote
inflammation and, ultimately, tumorigenesis. These data suggest a complex role of
autophagy in tumour formation.
As the morphology of necroptotic cell death is similar to that of necrosis, it has been
investigated whether pathological necrosis might actually represent, if only in part,
regulated necroptosis. Indeed, administration of necroptosis inhibitor necrostastin‑1
(Nec‑1) provides significant tissue protection and functional improvements in a range
of acute tissue injuries in vivo in mouse models (brain and heart ischaemia–reperfusion)
by mechanisms that are clearly distinct from the inhibition of pathological
apoptosis63,72,130. In particular, in a mouse model of stroke, the effect of Nec‑1 was both
temporally and mechanistically (measured through caspase‑3 activation) distinct from
that of apoptosis inhibitors. Furthermore, the protective effect of Nec‑1 did not require
co-administration of caspase inhibitors, although the pan-caspase inhibitor zVAD.fmk
and Nec‑1 did have an additive effect (FIG. 3).
Poly(ADP–ribose) polymerase‑1 (PARP1) inhibitors provide significant protective
effects in mouse brain and heart ischaemia–reperfusion injury, mouse models of
colitis and other inflammatory diseases, neurodegeneration and diabetes mellitus
(reviewed in Refs 131–133). PARP1 inhibitors have also emerged as promising
anti-cancer agents, increasing the sensitivity of resistant cancer cells to various
DNA-damaging agents and also selectively killing some tumour cells134.
Rapidly proliferating glycolytic cells might pose a sig-
nificant danger for the organism if they accumulate
DNA damage and, hence, have to be efficiently elimi-
nated; by contrast, vegetative cells can be allowed
more time to complete DNA repair. Along these lines,
PARP1 activation also leads to the specific release of the
inflammatory cytokine high mobility group protein‑B1
(HMGB1), which can alert immune cells to the presence
of dangerous cells with damaged DNA77. PARP1 also
mediates cell death that is induced by secondary DNA
damage associated with acute neuronal injury. In this
case, excitatory neuronal cell death leads to the trans-
location of the poly(ADP–ribose)-polymer into the
cytosol, triggering translocation of protein apoptosis-
inducing factor (AIF) from the mitochondria to the
nuclei, where it mediates cell death78,79. Interestingly,
DNA-damage-induced PARP1-mediated cell death
involves TNF receptor-associated factor-2 (TRAF2)–
RIP1-dependent activation of c-Jun N‑terminal
kinase‑1 (JNK1, also known as MAPK8), which con-
tributes to mitochondrial dysfunction and necrotic
death80. However, the relationship between this process
and necroptosis remains unclear.
Currently, it is difficult to make definitive conclu-
sions regarding the relationship of the two pathways
NLR of PARP1-induced cell death. However, it appears pos-
A mammalian protein that is
characterized by the presence
sible that NAD+ depletion and AIF activity act in the
of NACHT and Leu-rich repeat same pathway and that their relative contributions to
(LRR) domains. cell demise might depend on the specific cell milieu.
384 | May 2008 | volume 9 www.nature.com/reviews/molcellbio
© 2008 Nature Publishing Group
Although the fine details of PARP1-dependent cell death
remain to be sorted, it is clear that PARP1-mediated cell
death is an important subject for investigation because
of its role in various human pathologies (BOX 3).
The roots of regulated cell death
Curiously, although the phenomenon of apoptosis was
first described in the context of developmental regulation,
the function of apoptosis in development is not clear-
cut; C. elegans ced‑3 (loss-of-function) or ced‑4 (loss-of-
function) mutants are developmentally normal81. If PCD
does not provide a developmental advantage for nema-
todes that have it compared with ancient variants that
lacked it, what factors might have led to the selection and
evo­lution of PCD mechanisms? Interestingly, ced‑3 and
ced‑4 mutants were found to be significantly more sensi-
tive to death caused by Salmonella typhimurium infection
compared with wild-type worms82, raising the possibility
that the host defence response, rather than develop­
mental cell death, might be the primordial function
of apoptosis.
The host defence function of apoptosis has been
expanded in mammalian cells. A large family of mamma-
lian NLR (NOD-like receptor) proteins, homologous to 
C. elegans CED‑4 and mammalian APAF1, function
to regulate the activation of caspases in response to
intracellular pathogens83. NLRs contain three distinct
domains: an N‑terminal caspase-recruitment domain
(CARD) or pyrin effector domain (with the exception
of neuronal apoptosis-inhibitory protein (NAIP) and,
possibly, NOD5 (also known as NLRX1)); a nucleotide-
binding and oligomerization domain (the so-called
NACHT domain); and a variable number of C‑terminal
Leu-rich repeats (LRRs) (BOX 1). The 22 identified NLRs
can be divided into two large sub-classes — the NODs
(NOD1–5), which activate the RIP2–NF-κB pathway,
and the NLRs, consisting of NALP1–14 (NALP is named
after NACHT‑, LRR- and pyrin domain (PYD)-containing
proteins), IPAF (also known as NLRC4) and NAIP,
which promote caspase‑1 activation84. In addition, the
NLR protein CIITA (major histocompatibility complex
(MHC) class II trans-activator) serves the function of
master regulator of MHC class II transcription85. At least
some of the NALPs function by recruiting the adaptor
protein ASC through a homotypic PYD–PYD inter-
action, and ASC in turn recruits caspase‑1 through a
CARD–CARD interaction86. Oligomerization of NALPs
brings the inflammatory caspases, such as caspase‑1 and
caspase‑5, into close proximity to promote their activa-
tion. This NALP protein complex is called the inflam-
masome87. The activation of caspase‑1 in turn leads to
the processing and release of interleukin‑1β (IL-1β) and
IL‑18, which serve important pro-inflammatory func-
tions. The NALP1-mediated caspase‑1 activation is also
regulated by anti-apoptotic proteins of the BCL2 family88,
providing a possible role for BCL2 family proteins in
regulating the host defence response.
Interestingly, the origins of the NLR family can be
traced to non-apoptotic regulators in simple organisms,
potentially providing an exciting insight into the evo­
lutionary origins of mammalian apoptosis. Mammalian

PAMPs
Components of pathogens that
are recognized by the innate
immune system, including
lipopolysaccharide,
peptidoglycans and viral RNA.
DAMPS
Changes in the cellular
environment that are
associated with pathogenic
infection, such as an increase
in ATP concentration.
PIDDosome
A caspase‑2-activating
complex that is formed by the
multimerization of PIDD and
RAIDD proteins.
nature reviews | molecular cell biology volume 9 | May 2008 | 385
NLRs functionally and structurally resemble NLR-like
proteins, such as plant R-gene-encoded proteins89, that
are present in many primitive organisms (FIG. 2). The
R-gene-encoded proteins contain LRRs, which sense
intracellular pathogens and pathogen-induced danger sig-
nals and activate inflammatory signalling90. In a manner
that is highly analogous to the plant innate immune sys-
tem, mammalian inflammasomes recognize signals that
are presented by intracellular pathogens and mediate the
activation of death in infected cells (such as macrophages)
through caspase‑1-dependent apoptosis (pyroptosis) and
induction of inflammatory signalling through caspase‑1-
dependent processing of IL-1β, IL-18 (Ref. 5) and probably
IL-33 (Ref. 91).
Similar to hundreds of plant R-gene-encoded proteins,
distinct mammalian inflammasome complexes sense
particular conserved pathogen-associated molecular pat-
terns (PAMPs; these include components of the bacterial
cell wall, bacterial flagellin, and bacterial and viral RNA)
and pathogen-induced danger-associated molecular
patterns (DAMPs; these include changes in ATP levels and
intracellular potassium concentrations)89. For example,
the NALP1 inflammasome senses bacterial cell wall pepti­
doglycans92, whereas the IPAF inflammasome is activated
by cytosolic flagellin93,94. In addition, recent studies
showed that plant R-gene-encoded proteins can sense
PAMPs in the nuclei to induce transcriptional changes
that promote an immune response95. It is important to
determine whether this can also represent an additional
mode of action of mammalian NLRs. Curiously, whereas
the plant innate immune response can lead to cell death
at the infection site90, involving measurable induction of
caspase-like activity96, it is carried out by non-caspase
molecules, such as legumains. Plant proteins displaying
sequence homology and structure similarity to caspases,
termed metacaspases, probably represent a different
branch of the evolutionary tree97.
Based on these observations, the mammalian
inflammasome might be a convergence point between
a simple ced-gene-mediated apoptotic pathway and
the NLR-mediated innate immune pathway, providing
capabilities for PAMP or DAMP sensing by NLRs, as
well as caspase activation. Therefore, the mammalian
apoptotic machinery might have evolved by converging
the simple apoptotic machinery, such as that found in
C. elegans, with a separate and even more ancient and
highly conserved innate immune system. Furthermore,
because mammalian CED‑4-like factors (such as NLRs,
APAF1 and p53-induced protein with a death domain
(PIDD)) function similarly in caspase activation,
develop­ment of the mammalian apoptosis machinery
might have been primarily driven by the requirements
for a more efficient host defence system, rather than the
more complex homeostatic and developmental regula-
tion in higher eukaryotes. Although retaining the basic
layout of the PCD mechanism in C. elegans, the conver­
gence of this pathway with NLR regulation led to the
acquisition of a number of unique features that are
characteristic of mammalian apoptosis, including
direct regulation of the mammalian CED‑4 orthologues
by specific activators (such as cytochrome c, ATP and
© 2008 Nature Publishing Group
REVIEWS
a NLR PAMPs
DAMPs
SGT1 HSP90 (ATP)
LRR NACHT
Cell death
Pyrin Caspase-1
CARD Cytokine
RIP2
processing
Gene expression
b NB-LRR PAMPs (NODs)
DAMPs
SGT1 HSP90
LRR NB
Cell death
(hypersensitive response)
TIR
CC
Gene expression
Figure 2 | Mammalian NLR proteins and NB‑LRR proteins
from simpler organisms Nature
haveReviews
similar| Molecular
functions.Cell Biology
Although
NLR (panel a) and NB‑LRR (panel b) proteins (such as those
encoded by plant R-genes) have variable domain
architecture83,89,90, all NLR and NB‑LRR proteins are
characterized by the presence of: Leu-rich repeats (LRR),
which have significant variability (because of alternative
splicing136) and are involved in pathogen-associated
molecular pattern (PAMP) sensing; nucleotide-binding
ATPase oligomerization domains (NACHT or NB; these
make-up the ‘oligomerization domain’); and effector
domains (CARD and pyrin in NLRs; TIR and coiled coil (CC)
in NB‑LRRs), which are involved in the recruitment of
downstream factors, such as caspase‑1 or RIP2 in the case of
IPAF and NOD proteins, or additional adaptors, such as ASC,
in the case of NALP3 protein83. The mammalian NLRs
(panel a) also display multiple functional similarities with
NB‑LRRs (panel b) in that they function as part of the innate
immune response. Other similarities include: NLRs and
NB‑LRRs both require SGT1–HSP90 binding to maintain the
proteins in an inactive, but signal-competent state; they are
both activated by PAMPs and danger-associated molecular
patterns (DAMPs) in vivo and in vitro; and their cell death and
inflammatory responses are activated through effector
domains. These similarities suggest that mammalian NLR
proteins (and other APAF1-like molecules, such as APAF1
and PIDD) probably evolved from primitive NB‑LRR proteins.
ASC, apoptotic speck protein; IPAF, ICE-protease activating
factor; NB-LRR, nucleotide-binding Leu-rich-repeat; NLR,
nucleotide-binding and oligomerization domain (NOD)-like
receptor; NALP3, NACHT‑, LRR- and pyrin domain (PYD)-
containing protein-3; TIR, translocated intimin receptor.
patho­gens) and divergence of the developmental, geno-
toxic and inflammatory pathways, which are regulated by
separate sets of activating complexes (the apoptosome,
PIDDosome and inflammasome, respectively) (BOX 1).
Evolution of non-apoptotic cell death mechanisms
Whereas PARP1-mediated non-apoptotic cell death
probably evolved as part of the cellular response to
DNA damage, other non-apoptotic cell death mecha-
nisms might have evolved as a part of host defence
responses.
REVIEWS

a Predominantly necroptosis b Predominantly apoptosis One could speculate that autophagic cell death might have
arisen from the need to ensure the survival of the whole
organism through sacrificing infected cells. Both type I and
II interferons (IFNs) modulate macroautophagy as a part
of the antiviral response98. In this regard, it is interesting
that treatment of human HeLa cells with IFNγ leads to
the induction of autophagy through the activation of
death-associated protein (DAP) kinases104, and overactiv­
ation of DAP kinases can lead to autophagic cell death.
Thus, autophagic cell death might be part of the antiviral
No cytoprotection Maximal Maximal No cytoprotection response that is activated by IFNs.
cytoprotection cytoprotection The functional significance of necroptosis in the host
defence response is less clear. However, RIP1, a kinase
c Mixed model d Synergistic model
that is crucially involved in the activation of necroptosis,
has an important role mediating the activation of NF‑κB
and IFN genes in the context of the innate immune
system105, although apparently in a kinase-independent
manner. RIP1 signalling can be triggered by extracellular
pathogen-sensing Toll-like receptors106,107, components
of invading pathogens108 and inflammatory cytokines,
such as TNFα. The closest D. melanogaster homologue
of RIP1, IMD, is also a key mediator of the innate immune
response to Gram-negative bacteria, acting upstream of
Partial cytoprotection Partial cytoprotection No cytoprotection No cytoprotection a number of factors that are also linked to RIP1 signal-
ling in mammalian cells, including the D. melanogaster
homologues of FADD, caspase‑8, IKK (inhibitor of
nuclear factor (NF)-κB (IκB) kinase) complex and
NF‑κB109. Furthermore, IMD can also mediate caspase-
dependent apoptosis109, similar to the recently discovered
RIP1-dependent, caspase‑8-mediated apoptosis cascade
Maximal Maximal
cytoprotection cytoprotection in mammalian cells110.
Curiously, IMD lacks a kinase domain, so kinase-
zVAD.fmk Apoptotic cell Healthy cell dependent induction of necroptosis might be an evo-
Nec-1 Necroptotic cell lutionarily novel addition to the repertoire of RIP1
functions. Therefore, RIP1 might promote both the
Figure 3 | Plasticity of cell death activation in vivo. The existence of multiple cell
death mechanisms suggests that careful consideration should be given to determine induction of specific inflammatory signalling (by NF-κB
Nature Reviews | Molecular Cell Biology
which mechanism (or mechanisms) is primarily activated in any particular injury and IFN upregulation) and the elimination of infected
paradigm if therapeutic approaches are to prove useful. As an example, multiple cells through a pro-inflammatory process (necrosis). The
outcomes of anti-apoptotic or anti-necroptotic therapies can be anticipated depending promotion of necrosis can by itself serve to potentiate
on the specific injury paradigm. a | Predominant necrotic death might occur when the the antibacterial response by causing a leakage of cellular
endogenous conditions prohibit apoptosis. Use of necrostatin‑1 (Nec-1), which inhibits contents and the specific release of pro-inflammatory
RIP1-dependent necroptosis, might provide maximal cytoprotective benefit as a single mediators, such as IL-6 (Ref. 111). Recently discovered
agent under these circumstances. b | Alternatively, apoptotic cell death might be cell-death-independent activation of autophagy by
predominant in cell populations that are not subjected to excessive external stress, or TLR4–RIP1 as a potential mechanism for bacterial clear-
that are intrinsically deficient in necroptosis activation58,63. zVAD.fmk prevents apoptosis
ance also fits well with this notion103. These data suggest
by inhibiting caspases. c | A mixture of apoptotic and necroptotic cell death might occur,
leading to a significant, but partial, cytoprotective effect of each treatment and an that the evolution of the innate immune response might
additive effect of combination therapy. d | Apoptosis might be the predominant ‘primary’ have led to the acquisition of the RIP1-kinase-mediated
form of cell death. However, inhibition of apoptosis might result in the activation of necroptotic response by the IMD pathway.
necroptosis. In this scenario, neither apoptosis nor necroptosis inhibitors might work as
single agents, and combined treatment could provide maximal cytoprotective benefit. Opportunities for cytoprotective therapy
Catastrophic cell death is the main underlying cause of
death and lifelong disabilities in a broad range of human
Autophagy has a well-established role in defending diseases, from acute disorders (such as stroke, myo­cardial
against viral and bacterial invasion (reviewed in Ref. 98). infarction, brain and spinal cord trauma and septic shock)
Sindbis virus, a single-stranded RNA virus of the Togavirus to chronic neurodegenerative conditions. Cell death is
Macroautophagy family, causes encephalitis, which can be amelio­rated by also an important compounding factor as a side effect
The sequestration of cytosolic overexpression of Beclin-1 in transgenic mice99. Bacteria of chemotherapy, many inflammatory diseases, diabetes
components in that escape into the cytosol from the endosomes can be and other conditions. Therefore, the development of effi-
autophagosomes and their
subsequent degradation when
engulfed by macroautophagy100,101. Furthermore, autophagic cient strategies to inhibit pathological cell death remains
autophagosomes fuse with machinery promotes the clearance of extracellular a key challenge of cell death research and a crucial unmet
lysosomes. bacteria that are recognized by the TLR4 receptor102,103. medical need.

386 | May 2008 | volume 9 www.nature.com/reviews/molcellbio

© 2008 Nature Publishing Group
 REVIEWS

a Survival mode b Apoptosis mode c Necroptosis mode

TNFα TNFα TNFα

TNFR TNFR TNFR

Cell
TRAF2

TRAF2

TRAF2
membrane

Necroptosis complex
RIP1

RIP1

RIP1

RIP1

RIP1
RIP1
TRADD

TRADD

TRADD
Ub ? Ub ? Ub ?
Complex I

Ub Ub Ub
Ub Ub Ub
Rac1

Rac1

Rac1
Ub ? Ub ? Ub ?
DISC
NIK

NIK
IKK

IKK

IKK
NIK
TRADD

NADPH NADPH NADPH

RIP1

oxidase oxidase oxidase

FADD DISC DISC
FLIP
NF-κB Caspase-8 Caspase-8 NF-κB
Cycloheximide,
Low level ZFRA, FAK–/–
zVAD.fmk,
ischaemic insult,
Survival Apoptosis caspase-8–/– Necroptosis
Inflammation
Proliferation Figure 4 | Activation of alternative cell fates following TNFα stimulation. In many cell types, apoptosis is not the
default response to tumour-necrosis factor‑α (TNFα) stimulation — multiple cell fates can be independently adopted.
a | In many cases, activation of nuclear factor-κB (NF‑κB) signalling, resulting from NF‑κB-activating complex I formation
and RIP1 polyubiquitylation136, is a primary response to TNFα. In this scenario, RIP1 ubiquitylation limits the formation of
pro-apoptotic signalling complexes137. In addition, NF-κB transcriptionally upregulates the expression of pro-survival
genes137. The low level of caspase‑8 cleaves RIP1, which inhibits necroptosis. b | Activation of apoptosis as a primary
response to TNFα requires specific apoptosis-promoting conditions, such as the presence Nature Reviews
of protein| Molecular
synthesisCell Biology
inhibitors
(cycloheximide), the overexpression of zinc finger-like protein (ZFRA) polypeptide138 or a deficiency in focal adhesion
kinase (FAK) kinase signalling139. This leads to efficient pro-apoptotic death-inducing-signalling complex (DISC)
formation136 and caspase‑8 activation. c | Activation of necroptosis as a primary response to TNFα requires suppression
of apoptotic signalling or, at least, caspase activity (by caspase inhibitors). Rapid loss of ATP140–143, conditions of
excessive reactive oxygen or nitrogen species production144 or ischaemic conditions63,130 can provide environments
that are non-permissive to apoptosis. Notably, all three of these pathways represent independent cell fates that are
selected on the basis of the specifics of the cellular regulation. IKK, inhibitor of NF-κB (IκB) kinase; NIK, NF-κB-inducing
kinase; RIP1, receptor-interacting protein-1; TRADD, TNF receptor type 1-associated death domain protein;
TRAF2, TNF receptor-associated factor-2; Ub, ubiquitin.

Interferon
An inflammatory cytokine that
is produced by cells as part of
the innate immune response.
The discovery of apoptosis and the development of
specific genetic and small molecule methods to inhibit
pathological apoptosis in vivo have shown that pathological
cell death can, indeed, be targeted for therapeutic benefit.
However, the success of anti-apoptotic therapies has been
limited, perhaps because of our lack of understanding of
the complexity of cell death regulation in mammalian
cells. The appreciation of such complexity leads us to sug-
gest that when considering the possibility of inhibiting a
specific pathological mechanism of cell death, we must
consider several issues. Does one particular form of cell
death have a major role in the injury (FIG. 3)? Alternatively,
are several cell death mechanisms operational in the
injured tissue? If several mechanisms are operational,
then combination therapy might provide maximal benefit.
Whether a combination therapy will be effective depends
on the contribution of each form of death, not only to the
tissue injury, but also to the functional decline of the tissue
(FIG. 3c). We must also consider whether a possible backup
cell death mechanism that might be activated in the event
of the primary mechanism is being inhibited. Given these
possibilities, cytoprotective treatment might not only be
improved by combination treatment, it might require
combination treatment (FIG. 3d).
It is important to keep in mind that although apopto-
sis may be the preferred type of physiological cell death,
the option to die by apoptosis might not always be avail-
able under in vivo conditions. Situations that involve an
imbalance of ROS-generation and ROS-detoxification,
limited energy metabolism or a lack of proper protein
synthesis might restrict the ability of cells to activate
apoptotic cell death. Under such circumstances, cells
might choose to die through one of the alternative
cell death pathways. The existence of other cell death
options suggests that there might be some plasticity in
the choice of cell death programmes, with apoptosis
being only one of the spectrum of available regulated cell
death options. Furthermore, rather than considering a
hierarchal regulation of multiple cell death mechanisms,
in which cells first activate apoptosis and only undergo
non-apoptotic cell death if apoptosis is inhibited, we
propose that commitment to apoptosis might not even
be necessary for the activation of non-apoptotic cell
demise. In other words, non-apoptotic signalling can
be initiated independently through alternative mecha-
nisms to carry out the order of cellular execution under
conditions that are ill-suited for apoptosis, but ideal for
non-apoptotic cell death (FIG. 4).

nature reviews | molecular cell biology volume 9 | May 2008 | 387

© 2008 Nature Publishing Group
REVIEWS

The discovery of the apoptotic programme opened the
door for the development of specific cell-death-targeting
‘smart’ therapies. Pathological cell death pro­cesses might
represent a ‘conscious’ decision of the cell in response
to specific pathological signals, which would allow the
development of specific approaches to influence this
‘choice’ by small molecule or protein-based agents that
could target the apoptotic signalling machinery. This
has already led to the development of multiple classes
of agent, such as BCL2 and inhibitor of apoptosis (IAP)
proteins, that specifically trigger apoptosis in cancer
cells112. Although these agents are still in the early stages
of clinical development, preliminary evidence is promis-
ing. At the same time, therapies to eliminate catastrophic
tissue damage and functional decline, which are asso-
ciated with pathological cell death in various human
pathologies (from stroke to myocardial infarction), are
still limited. The recent discovery of regulated non-
apoptotic cell death might offer a new hope for treating
these diseases. Although the study of these forms of cell
death is still in its infancy, a number of promising results
have already been generated (BOX 3).
Conclusion
Less than 2 decades ago, cell death was categorically con-
sidered to be passive and uninteresting. The discovery
of mammalian homologues of C. elegans cell death genes
led to the understanding that cell death, in the form of
apoptosis, can be a highly regulated cellular mechanism.
In-depth characterization of mammalian apoptosis
uncovered both conservation of the basic layout of apop-
totic signalling in C. elegans and evolutionary expansion
of the protein families of apoptotic regulators. Moreover,
these features allowed specialized activation of cell death
responses by various upstream stimuli, improved inte-
gration of apoptosis with other cellular signalling and
metabolic pathways, and increased fidelity of apoptosis
execution, due to the redundancy in the functions of
individual members of apoptosis regulatory families.
Recent characterization of the inflammasome pathway
of caspase‑1 activation revealed that the evolution of
mammalian apoptosis probably involved convergence
of the primitive apoptosis machinery with the innate
immune system.
In the past few years we have also begun to appreciate
that apoptosis is not the only form of regulated cell death.
Three of the best-understood examples of non-apoptotic
cell death are type II cell death, necroptosis and PARP1-
mediated necrotic death (see above). Although these
processes can serve functions that are complimentary
or reinforce apoptosis, it is likely that they have evolved
to serve specific non-redundant functions in responses to
pathogen infection, nutrient and energy deprivation,
and DNA damage. The changing perception of regu-
lated cell death as an array of diverse responses, rather
than a single apoptotic pathway, implicates complexity
and provides novel opportunities for cytoprotective
therapies. In particular, the discovery of the specific
regulated, morphologically necrotic, non-apoptotic cell
death mechanisms suggests that at least a subset of
necrotic pathological cell death might also be regulated
by cellular mechanisms and, therefore, could be amend-
able to therapeutic drug development. Understanding
how cell death operates under the specific conditions of
particular human diseases might bring in a new era
of cytoprotective drug development.

1. Yuan, J. & Horvitz, H. R. A first insight into the
molecular mechanisms of apoptosis. Cell 116,
S53–S56 (2004).
The authors provide in-depth insight into the
discovery of the apoptotic machinery in C. elegans.
2. Clarke, P. G. Developmental cell death: morphological
diversity and multiple mechanisms. Anat. Embryol.
181, 195–213 (1990).
This paper provides an early morphological
analysis of developmental cell death and describes
the existence of multiple forms of PCD.
3. Twomey, C. & McCarthy, J. V. Pathways of apoptosis
and importance in development. J. Cell. Mol. Med. 9,
345–359 (2005).
4. Yoshida, H. et al. Apaf1 is required for mitochondrial
pathways of apoptosis and brain development. Cell
94, 739–750 (1998).
5. Gu, Y. et al. Activation of interferon-γ inducing factor
mediated by interleukin-1β converting enzyme.
Science 275, 206–209 (1997).
6. Wei, M. C. et al. tBID, a membrane-targeted death
ligand, oligomerizes BAK to release cytochrome c.
Genes Dev. 14, 2060–2071 (2000).
7. McConkey, D. J. Biochemical determinants of
apoptosis and necrosis. Toxicol. Lett. 99, 157–168
(1998).
8. Zong, W. X. & Thompson, C. B. Necrotic death as a cell
fate. Genes Dev. 20, 1–15 (2006).
9. Horvitz, H. R., Shaham, S. & Hengartner, M. O. The
genetics of programmed cell death in the nematode
Caenorhabditis elegans. Cold Spring Harb. Symp.
Quant. Biol. 59, 377–385 (1994).
10. Conradt, B. & Xue, D. Programmed cell death.
WormBook, 1–13 (2005).
11. Metzstein, M. M., Stanfield, G. M. & Horvitz, H. R.
Genetics of programmed cell death in C. elegans: past,
present and future. Trends Genet. 14, 410–416
(1998).
12. Hofmann, E. R. et al. Caenorhabditis elegans HUS‑1
is a DNA damage checkpoint protein required for
genome stability and EGL‑1‑mediated apoptosis.
Curr. Biol. 12, 1908–1918 (2002).
13. Tait, S. W. et al. Apoptosis induction by Bid requires
unconventional ubiquitination and degradation of its
N‑terminal fragment. J. Cell Biol. 179, 1453–1466
(2007).
14. Konishi, Y., Lehtinen, M., Donovan, N. & Bonni, A.
Cdc2 phosphorylation of BAD links the cell cycle to the
cell death machinery. Mol. Cell 9, 1005–1016 (2002).
15. Zha, J., Weiler, S., Oh, K. J., Wei, M. C. & Korsmeyer,
S. J. Posttranslational N‑myristoylation of BID as a
molecular switch for targeting mitochondria and
apoptosis. Science 290, 1761–1765 (2000).
16. Li, H., Zhu, H., Xu, C. J. & Yuan, J. Cleavage of BID by
caspase 8 mediates the mitochondrial damage in the
Fas pathway of apoptosis. Cell 94, 491–501 (1998).
17. Youle, R. J. & Strasser, A. The BCL‑2 protein family:
opposing activities that mediate cell death. Nature
Rev. Mol. Cell Biol. 9, 47–59 (2008).
18. Scorrano, L. & Korsmeyer, S. J. Mechanisms of
cytochrome c release by proapoptotic BCL‑2 family
members. Biochem. Biophys. Res. Commun. 304,
437–444 (2003).
This paper describes in detail the molecular
mechanism of the key step in apoptosis where
mitochondrial cytochrome c is released.
19. Willis, S. N. et al. Apoptosis initiated when BH3
ligands engage multiple Bcl‑2 homologs, not Bax or
Bak. Science 315, 856–859 (2007).
This paper presents evidence that suggests that
anti-apoptotic BCL2 family members, rather than
BAX and BAK, are the targets of BH3-only proteins.
20. Gogvadze, V., Orrenius, S. & Zhivotovsky, B. Multiple
pathways of cytochrome c release from mitochondria
in apoptosis. Biochim. Biophys. Acta 1757, 639–647
(2006).
21. Degterev, A., Boyce, M. & Yuan, J. A decade of
caspases. Oncogene 22, 8543–8567 (2003).
22. Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S.
& Peter, M. E. Apoptosis signaling by death receptors.
Eur. J. Biochem. 254, 439–459 (1998).
23. Micheau, O. & Tschopp, J. Induction of TNF receptor
I‑mediated apoptosis via two sequential signaling
complexes. Cell 114, 181–190 (2003).
In this paper, upstream signalling events that lead
to the activation of apoptosis are described.
24. Barnhart, B. C., Alappat, E. C. & Peter, M. E. The
CD95 type I/type II model. Semin. Immunol. 15,
185–193 (2003).
In this paper, distinct pathways (direct or
mitochondria-mediated) of executioner caspase
activation by death receptors are described in detail.
25. McStay, G. P., Salvesen, G. S. & Green, D. R.
Overlapping cleavage motif selectivity of caspases:
implications for analysis of apoptotic pathways. Cell
Death Differ. 15, 322–331 (2007).
26. Sprick, M. R. et al. Caspase‑10 is recruited to and
activated at the native TRAIL and CD95 death-
inducing signalling complexes in a FADD-dependent
manner but can not functionally substitute caspase‑8.
EMBO J. 21, 4520–4530 (2002).
27. Su, H. et al. Requirement for caspase‑8 in NF‑kB
activation by antigen receptor. Science 307,
1465–1468 (2005).
28. Lassus, P., Opitz-Araya, X. & Lazebnik, Y. Requirement
for caspase‑2 in stress-induced apoptosis before
mitochondrial permeabilization. Science 297,
1352–1354 (2002).
In this paper, a specific role for caspase‑2 in
genotoxic stress-induced apoptosis is shown.
29. Hitomi, J. et al. Involvement of caspase‑4 in
endoplasmic reticulum stress-induced apoptosis and
Aβ-induced cell death. J. Cell Biol. 165, 347–356
(2004).

388 | May 2008 | volume 9 www.nature.com/reviews/molcellbio

© 2008 Nature Publishing Group

30. Nakagawa, T. et al. Caspase‑12 mediates
endoplasmic‑reticulum‑specific apoptosis and
cytotoxicity by amyloid-β. Nature 403, 98–103 (2000).
This paper provides the first demonstration of the
specific role of caspase‑12 in ER stress response.
31. Zou, H., Li, Y., Liu, X. & Wang, X. An
APAF‑1cytochrome c multimeric complex is a
functional apoptosome that activates procaspase‑9.
J. Biol. Chem. 274, 11549–11556 (1999).
The authors establish the existence of the
caspase‑9‑activating apoptotosome complex.
32. Gao, Z., Shao, Y. & Jiang, X. Essential roles of the
Bcl‑2 family of proteins in caspase‑2‑induced
apoptosis. J. Biol. Chem. 280, 38271–38275 (2005).
33. Bouillet, P. et al. Proapoptotic Bcl‑2 relative Bim
required for certain apoptotic responses, leukocyte
homeostasis, and to preclude autoimmunity. Science
286, 1735–1738 (1999).
This is one of the earliest papers establishing
specific roles of a particular BH3-only factor, BIM,
in response to specific upstream signals.
34. Yu, J. & Zhang, L. The transcriptional targets of p53 in
apoptosis control. Biochem. Biophys. Res. Commun.
331, 851–588 (2005).
35. Schmelzle, T. et al. Functional role and oncogene-
regulated expression of the BH3-only factor Bmf in
mammary epithelial anoikis and morphogenesis.
Proc. Natl Acad. Sci. USA 104, 3787–3792 (2007).
36. Chen, L. et al. Differential targeting of prosurvival
Bcl‑2 proteins by their BH3-only ligands allows
complementary apoptotic function. Mol. Cell 17,
393–403 (2005).
This paper demonstrates that selective interactions
of pro- and anti-apoptotic BCL2 family members
contribute to the regulation of the mitochondrial
step in apoptosis.
37. Takai, Y. et al. Caspase‑12 compensates for lack of
caspase‑2 and caspase‑3 in female germ cells.
Apoptosis 12, 791–800 (2007).
38. Troy, C. M. et al. Death in the balance: alternative
participation of the caspase‑2 and ‑9 pathways in
neuronal death induced by nerve growth factor
deprivation. J. Neurosci. 21, 5007–5016 (2001).
39. Garcia-Calvo, M. et al. Inhibition of human caspases
by peptide-based and macromolecular inhibitors.
J. Biol. Chem. 273, 32608–32613 (1998).
40. Abraham, M. C., Lu, Y. & Shaham, S.
A morphologically conserved nonapoptotic program
promotes linker cell death in Caenorhabditis elegans.
Dev. Cell 12, 73–86 (2007).
41. Oppenheim, R. W. et al. Programmed cell death of
developing mammalian neurons after genetic deletion
of caspases. J. Neurosci. 21, 4752–4760 (2001).
42. Yuan, J. Inducing autophagy harmlessly. Autophagy 4,
249–250 (2007).
43. Lee, C. Y. & Baehrecke, E. H. Steroid regulation of
autophagic programmed cell death during
development. Development 128, 1443–1455
(2001).
44. Berry, D. L. & Baehrecke, E. H. Growth arrest and
autophagy are required for salivary gland cell
degradation in Drosophila. Cell 131, 1137–1148
(2007).
This paper demonstrates that autophagic cell
death is specifically activated during development
under apoptosis-competent conditions.
45. Marino, G. et al. Tissue-specific autophagy alterations
and increased tumorigenesis in mice deficient in
Atg4C/autophagin‑3. J. Biol. Chem. 282,
18573–18583 (2007).
46. Komatsu, M. et al. Impairment of starvation-induced
and constitutive autophagy in Atg7-deficient mice.
J. Cell Biol. 169, 425–434 (2005).
47. Kuma, A. et al. The role of autophagy during the early
neonatal starvation period. Nature 432, 1032–1036
(2004).
This paper demonstrates the crucial role of
autophagy in vivo in maintaining survival under
nutrient-deprivation conditions.
48. Juhasz, G., Erdi, B., Sass, M. & Neufeld, T. P.
Atg7-dependent autophagy promotes neuronal
health, stress tolerance, and longevity but is
dispensable for metamorphosis in Drosophila. Genes
Dev. 21, 3061–3066 (2007).
49. Espert, L. et al. Autophagy is involved in T cell death
after binding of HIV‑1 envelope proteins to CXCR4.
J. Clin. Invest. 116, 2161–2172 (2006).
50. Pattingre, S., Espert, L., Biard-Piechaczyk, M. &
Codogno, P. Regulation of macroautophagy by mTOR
and Beclin 1 complexes. Biochimie 90,
313–323(2007).
nature reviews | molecular cell biology volume 9 | May 2008 | 389
51. Maiuri, M. C., Zalckvar, E., Kimchi, A. & Kroemer, G.
Self-eating and self-killing: crosstalk between
autophagy and apoptosis. Nature Rev. Mol. Cell Biol.
8, 741–752 (2007).
52. Ullman, E. et al. Autophagy promotes necrosis in
apoptosis-deficient cells in response to ER stress. Cell
Death Differ. 15, 422–425 (2008).
53. Shimizu, S. et al. Role of Bcl‑2 family proteins in a non-
apoptotic programmed cell death dependent on
autophagy genes. Nature Cell Biol. 6, 1221–1228
(2004).
54. Oberstein, A., Jeffrey, P. D. & Shi, Y. Crystal structure
of the Bcl‑XL‑Beclin 1 peptide complex: Beclin 1 is a
novel BH3-only protein. J. Biol. Chem. 282,
13123–13132 (2007).
55. Pattingre, S. et al. Bcl‑2 antiapoptotic proteins inhibit
Beclin 1‑dependent autophagy. Cell 122, 927–939
(2005).
This paper presents evidence for the regulation of
autophagy by the anti-apoptotic BCL2 family
members, establishing convergent regulation of
apoptosis and autophagy.
56. Tracy, K. & Macleod, K. F. Regulation of mitochondrial
integrity, autophagy and cell survival by BNIP3.
Autophagy 3, 616–619 (2007).
57. Rashmi, R., Pillai, S. G., Vijayalingam, S., Ryerse, J. &
Chinnadurai, G. BH3-only protein BIK induces
caspase-independent cell death with autophagic
features in Bcl‑2 null cells. Oncogene 27, 1366–1375
(2007).
58. Khwaja, A. & Tatton, L. Resistance to the cytotoxic
effects of tumor necrosis factor a can be overcome
by inhibition of a FADD/caspase-dependent signaling
pathway. J. Biol. Chem. 274, 36817–36823
(1999).
59. Matsumura, H. et al. Necrotic death pathway in Fas
receptor. J. Cell Biol. 151, 1247–1256 (2000).
60. Vercammen, D. et al. Inhibition of caspases increases
the sensitivity of L929 cells to necrosis mediated by
tumor necrosis factor. J. Exp. Med. 187, 1477–1485
(1998).
61. Vercammen, D. et al. Dual signaling of the Fas
receptor: initiation of both apoptotic and necrotic cell
death pathways. J. Exp. Med. 188, 919–930 (1998).
62. Chan, F. K. et al. A role for tumor necrosis factor
receptor‑2 and receptor-interacting protein in
programmed necrosis and antiviral responses. J. Biol.
Chem. 278, 51613–51621 (2003).
63. Degterev, A. et al. Chemical inhibitor of nonapoptotic
cell death with therapeutic potential for ischemic brain
injury. Nature Chem. Biol. 1, 112–119 (2005).
This paper describes a first‑in‑class potent and
selective inhibitor of necroptosis.
64. Holler, N. et al. Fas triggers an alternative,
caspase‑8‑independent cell death pathway using the
kinase RIP as effector molecule. Nature Immunol. 1,
489–495 (2000).
In this paper, the crucial role of RIP1 kinase activity
in the activation of necroptosis is first
demonstrated.
65. Kawahara, A., Ohsawa, Y., Matsumura, H., Uchiyama, Y.
& Nagata, S. Caspase-independent cell killing by Fas-
associated protein with death domain. J. Cell Biol.
143, 1353–1360 (1998).
This paper is one of the initial reports showing
induction of necrotic death by death-domain
receptor signals.
66. Festjens, N., Vanden Berghe, T. & Vandenabeele, P.
Necrosis, a well-orchestrated form of cell demise:
signalling cascades, important mediators and
concomitant immune response. Biochim. Biophys.
Acta 1757, 1371–1387 (2006).
This paper provides an in-depth analysis of the
signalling and execution pathways of regulated
necrosis.
67. Zheng, L. et al. Competitive control of independent
programs of tumor necrosis factor receptor-induced
cell death by TRADD and RIP1. Mol. Cell Biol. 26,
3505–3513 (2006).
This paper shows that apoptotic and necroptotic
signalling pathways diverge at the level of the
death-domain receptor.
68. Wang, K. et al. Structure-activity relationship analysis
of a novel necroptosis inhibitor, Necrostatin‑5.
Bioorg. Med. Chem. Lett. 17, 1455–1465 (2007).
69. Jagtap, P. G. et al. Structure-activity relationship study
of tricyclic necroptosis inhibitors. J. Med. Chem. 50,
1886–1895 (2007).
70. Teng, X. et al. Structure-activity relationship study of
novel necroptosis inhibitors. Bioorg. Med. Chem. Lett.
15, 5039–5044 (2005).
© 2008 Nature Publishing Group
REVIEWS
71. Temkin, V., Huang, Q., Liu, H., Osada, H. & Pope,
R. M. Inhibition of ADP/ATP exchange in receptor-
interacting protein-mediated necrosis. Mol. Cell Biol.
26, 2215–2225 (2006).
72. Lim, S. Y., Davidson, S. M., Mocanu, M. M.,
Yellon, D. M. & Smith, C. C. The cardioprotective effect
of necrostatin requires the cyclophilin‑D component of
the mitochondrial permeability transition pore.
Cardiovasc. Drugs Ther. 21, 467–469 (2007).
73. Suzuki, S. et al. Nur77 as a survival factor in tumor
necrosis factor signaling. Proc. Natl Acad. Sci. USA
100, 8276–8280 (2003).
74. Jagtap, P. & Szabo, C. Poly(ADP-ribose) polymerase
and the therapeutic effects of its inhibitors. Nature
Rev. Drug Discov. 4, 421–440 (2005).
75. Oei, S. L., Keil, C. & Ziegler, M. Poly(ADP-ribosylation)
and genomic stability. Biochem. Cell Biol. 83,
263–269 (2005).
76. Zong, W. X., Ditsworth, D., Bauer, D. E., Wang, Z. Q. &
Thompson, C. B. Alkylating DNA damage stimulates a
regulated form of necrotic cell death. Genes Dev. 18,
1272–1282 (2004).
This paper establishes the existence of the ‘death
by energy collapse’ mechanism of
PARP‑1‑mediated cell death.
77. Ditsworth, D., Zong, W. X. & Thompson, C. B.
Activation of poly(ADP)-ribose polymerase (PARP‑1)
induces release of the pro-inflammatory mediator
HMGB1 from the nucleus. J. Biol. Chem. 282,
17845–17854 (2007).
78. Andrabi, S. A. et al. Poly(ADP-ribose) (PAR) polymer is
a death signal. Proc. Natl Acad. Sci. USA 103,
18308–18313 (2006).
79. Yu, S. W. et al. Apoptosis-inducing factor mediates
poly(ADP-ribose) (PAR) polymer-induced cell death.
Proc. Natl Acad. Sci. USA 103, 18314–18319
(2006).
This paper describes the mechanism of the
PARP1–AIF cell death.
80. Xu, Y., Huang, S., Liu, Z. G. & Han, J. Poly(ADP-ribose)
polymerase‑1 signaling to mitochondria in necrotic cell
death requires RIP1/TRAF2-mediated JNK1
activation. J. Biol. Chem. 281, 8788–8795
(2006).
81. Ellis, H. M. & Horvitz, H. R. Genetic control of
programmed cell death in the nematode C. elegans.
Cell 44, 817–829 (1986).
82. Aballay, A. & Ausubel, F. M. Programmed cell death
mediated by ced‑3 and ced‑4 protects Caenorhabditis
elegans from Salmonella typhimurium-mediated
killing. Proc. Natl Acad. Sci. USA 98, 2735–2739
(2001).
83. Martinon, F., Gaide, O., Petrilli, V., Mayor, A. &
Tschopp, J. NALP inflammasomes: a central role in
innate immunity. Semin. Immunopathol. 29, 213–229
(2007).
This paper provides an extensive review of the
mechanism of action and regulation of mammalian
inflammasomes.
84. Martinon, F. & Tschopp, J. NLRs join TLRs as innate
sensors of pathogens. Trends Immunol. 26, 447–454
(2005).
85. Ting, J. P., Kastner, D. L. & Hoffman, H. M.
CATERPILLERs, pyrin and hereditary immunological
disorders. Nature Rev. Immunol. 6, 183–195
(2006).
86. Srinivasula, S. M. et al. The PYRIN-CARD protein ASC
is an activating adaptor for caspase‑1. J. Biol. Chem.
277, 21119–21122 (2002).
87. Martinon, F., Burns, K. & Tschopp, J. The
inflammasome: a molecular platform triggering
activation of inflammatory caspases and processing of
proIL-β. Mol. Cell 10, 417–426 (2002).
This paper provides an initial report demonstrating
the existance of the caspase‑1 activating
inflammasome complex.
88. Bruey, J. M. et al. Bcl‑2 and Bcl-XL regulate
proinflammatory caspase‑1 activation by interaction
with NALP1. Cell 129, 45–56 (2007).
This paper presents evidence that inflammasome
activity is inhibited by BCL2 family members.
89. Petrilli, V., Dostert, C., Muruve, D. A. & Tschopp, J.
The inflammasome: a danger sensing complex
triggering innate immunity. Curr. Opin. Immunol. 19,
615–622 (2007).
90. Jones, J. D. & Dangl, J. L. The plant immune system.
Nature 444, 323–329 (2006).
91. Schmitz, J. et al. IL‑33, an interleukin‑1‑like cytokine
that signals via the IL‑1 receptor-related protein ST2
and induces T helper type 2-associated cytokines.
Immunity 23, 479–490 (2005).
REVIEWS
92. Faustin, B. et al. Reconstituted NALP1 inflammasome
reveals two-step mechanism of caspase‑1 activation.
Mol. Cell 25, 713–724 (2007).
93. Franchi, L. et al. Cytosolic flagellin requires Ipaf for
activation of caspase‑1 and interleukin 1β in
salmonella-infected macrophages. Nature Immunol. 7,
576–582 (2006).
94. Miao, E. A. et al. Cytoplasmic flagellin activates
caspase‑1 and secretion of interleukin 1β via Ipaf.
Nature Immunol. 7, 569–575 (2006).
References 93 and 94 establish that IPAF
inflammasomes can sense intracellular PAMP
signals.
95. Shen, Q. H. et al. Nuclear activity of MLA immune
receptors links isolate-specific and basal disease-
resistance responses. Science 315, 1098–1103
(2007).
96. Woltering, E. J. Death proteases come alive. Trends
Plant Sci. 9, 469–472 (2004).
97. Vercammen, D., Declercq, W., Vandenabeele, P. &
Van Breusegem, F. Are metacaspases caspases?
J. Cell Biol. 179, 375–380 (2007).
98. Schmid, D. & Munz, C. Innate and adaptive immunity
through autophagy. Immunity 27, 11–21 (2007).
Reference 98 provides a detailed overview of the
role of autophagy in the immune regulation.
99. Liang, X. H. et al. Protection against fatal Sindbis virus
encephalitis by beclin, a novel Bcl‑2‑interacting
protein. J. Virol. 72, 8586–8596 (1998).
100. Nakagawa, I. et al. Autophagy defends cells against
invading group A Streptococcus. Science 306,
1037–1040 (2004).
This paper presents evidence that the activation of
autophagy can serve a host defence function.
101. Py, B. F., Lipinski, M. M. & Yuan, J. Autophagy limits
Listeria monocytogenes intracellular growth in the
early phase of primary infection. Autophagy 3,
117–125 (2007).
102. Sanjuan, M. A. et al. Toll-like receptor signalling in
macrophages links the autophagy pathway to
phagocytosis. Nature 450, 1253–1257 (2007).
103. Xu, Y. et al. Toll-like receptor 4 is a sensor for autophagy
associated with innate immunity. Immunity 27,
135–144 (2007).
104. Inbal, B., Bialik, S., Sabanay, I., Shani, G. & Kimchi, A.
DAP kinase and DRP‑1 mediate membrane blebbing
and the formation of autophagic vesicles during
programmed cell death. J. Cell Biol. 157, 455–468
(2002).
105. Meylan, E. & Tschopp, J. The RIP kinases: crucial
integrators of cellular stress. Trends Biochem. Sci. 30,
151–159 (2005).
A detailed review that discusses the structure and
function of the RIP kinase family.
106. Cusson-Hermance, N., Khurana, S., Lee, T. H.,
Fitzgerald, K. A. & Kelliher, M. A. Rip1 mediates the
Trif-dependent toll-like receptor 3‑ and 4‑induced
NF‑κB activation but does not contribute to interferon
regulatory factor 3 activation. J. Biol. Chem. 280,
36560–36566 (2005).
107. Meylan, E. et al. RIP1 is an essential mediator of Toll-
like receptor 3‑induced NF‑κB activation. Nature
Immunol. 5, 503–507 (2004).
108. Balachandran, S., Thomas, E. & Barber, G. N. A FADD-
dependent innate immune mechanism in mammalian
cells. Nature 432, 401–405 (2004).
109. Georgel, P. et al. Drosophila immune deficiency (IMD)
is a death domain protein that activates antibacterial
defense and can promote apoptosis. Dev. Cell 1,
503–514 (2001).
110. Petersen, S. L. et al. Autocrine TNFα signaling
renders human cancer cells susceptible to
Smac‑mimetic‑induced apoptosis. Cancer Cell 12,
445–456 (2007).
111. Vanden Berghe, T. et al. Necrosis is associated with
IL‑6 production but apoptosis is not. Cell Signal 18,
328–335 (2006).
112. Korkina, O. & Degterev, A. in Wiley Encyclopedia of
Chemical Biology (ed. Begley, T. P.) (John Wiley and
Sons, Ltd, 2008).
390 | May 2008 | volume 9 www.nature.com/reviews/molcellbio
113. Shimizu, S., Konishi, A., Kodama, T. & Tsujimoto, Y.
BH4 domain of antiapoptotic Bcl‑2 family members
closes voltage-dependent anion channel and inhibits
apoptotic mitochondrial changes and cell death. Proc.
Natl Acad. Sci. USA 97, 3100–3105 (2000).
114. Cheng, E. H. et al. Conversion of Bcl‑2 to a Bax-like
death effector by caspases. Science 278, 1966–1968
(1997).
115. Acehan, D. et al. Three-dimensional structure of the
apoptosome: implications for assembly, procaspase‑9
binding, and activation. Mol. Cell 9, 423–432 (2002).
This is the first paper to provide structural insight
into the functioning of the apoptosome.
116. Park, H. H. et al. Death domain assembly mechanism
revealed by crystal structure of the oligomeric
PIDDosome core complex. Cell 128, 533–546 (2007).
117. Mizushima, N. Autophagy: process and function.
Genes Dev. 21, 2861–2873 (2007).
This is an in-depth review that describes recent
progress in understanding autophagic regulation.
118. Yu, L. et al. Regulation of an ATG7–beclin 1 program
of autophagic cell death by caspase‑8. Science 304,
1500–1502 (2004).
This paper demonstrates activation of autophagic
cell death resulting from caspase inhibition.
119. Kim, Y. S., Morgan, M. J., Choksi, S. & Liu, Z. G.
TNF-induced activation of the Nox1 NADPH oxidase
and its role in the induction of necrotic cell death. Mol.
Cell 26, 675–687 (2007).
120. Schulze-Osthoff, K. et al. Cytotoxic activity of tumor
necrosis factor is mediated by early damage of
mitochondrial functions. Evidence for the involvement
of mitochondrial radical generation. J. Biol. Chem.
267, 5317–5323 (1992).
121. Festjens, N. et al. Butylated hydroxyanisole is more
than a reactive oxygen species scavenger. Cell Death
Differ. 13, 166–169 (2006).
122. Thon, L. et al. Ceramide mediates caspase-
independent programmed cell death. FASEB. J. 19,
1945–1956 (2005).
123. Ame, J. C., Spenlehauer, C. & de Murcia, G. The PARP
superfamily. Bioessays 26, 882–893 (2004).
This is a detailed review of the structure and
function of PARP family members.
124. Vahsen, N. et al. AIF deficiency compromises oxidative
phosphorylation. EMBO J. 23, 4679–4689 (2004).
In this report, the metabolic function of AIF in the
regulating activity of mitochondrial complex I is
established.
125. Zhu, H. et al. Cardiac autophagy is a maladaptive
response to hemodynamic stress. J. Clin. Invest. 117,
1782–1793 (2007).
126. Matsui, Y. et al. Distinct roles of autophagy in the
heart during ischemia and reperfusion: roles of AMP-
activated protein kinase and Beclin 1 in mediating
autophagy. Circ. Res. 100, 914–922 (2007).
127. Amaravadi, R. K. et al. Autophagy inhibition enhances
therapy-induced apoptosis in a Myc-induced model of
lymphoma. J. Clin. Invest. 117, 326–336 (2007).
This paper demonstrates that the induction of
autophagy in cancer cells in vivo enhances tumour
growth through the inhibition of apoptosis.
128. Edinger, A. L. & Thompson, C. B. Defective autophagy
leads to cancer. Cancer Cell 4, 422–424 (2003).
129. Degenhardt, K. et al. Autophagy promotes tumor cell
survival and restricts necrosis, inflammation, and
tumorigenesis. Cancer Cell 10, 51–64 (2006).
This paper demonstrates the plasticity of cancer
cell death activation under hypoxic conditions by
demonstrating the activation of apoptosis,
autophagy and necrosis that is dependent on the
expression of specific protein factors in the cell.
130. Smith, C. C. et al. Necrostatin: a potentially novel
cardioprotective agent? Cardiovasc. Drugs Ther. 21,
227–233 (2007).
131. de la Lastra, C. A., Villegas, I. & Sanchez-Fidalgo, S.
Poly(ADP-ribose) polymerase inhibitors: new
pharmacological functions and potential clinical
implications. Curr. Pharm. Des. 13, 933–962
(2007).
© 2008 Nature Publishing Group
132. Horvath, E. M. & Szabo, C. Poly(ADP-ribose)
polymerase as a drug target for cardiovascular disease
and cancer: an update. Drug News Perspect. 20,
171–181 (2007).
133. Kauppinen, T. M. & Swanson, R. A. The role of
poly(ADP-ribose) polymerase‑1 in CNS disease.
Neuroscience 145, 1267–1272 (2007).
134. Zaremba, T. & Curtin, N. J. PARP inhibitor
development for systemic cancer targeting. Anticancer
Agents Med. Chem. 7, 515–523 (2007).
135. Martinon, F. & Tschopp, J. Inflammatory caspases and
inflammasomes: master switches of inflammation. Cell
Death Differ. 14, 10–22 (2007).
136. Muppidi, J. R., Tschopp, J. & Siegel, R. M. Life and
death decisions: secondary complexes and lipid rafts
in TNF receptor family signal transduction. Immunity
21, 461–465 (2004).
137. O’Donnell, M. A., Legarda-Addison, D., Skountzos, P.,
Yeh, W. C. & Ting, A. T. Ubiquitination of RIP1
regulates an NF‑κB‑independent cell-death switch in
TNF signaling. Curr. Biol. 17, 418–424 (2007).
138. Hong, Q. et al. Zfra affects TNF-mediated cell death by
interacting with death domain protein TRADD and
negatively regulates the activation of NF‑κB, JNK1,
p53 and WOX1 during stress response. BMC Mol.
Biol. 8, 50 (2007).
139. Takahashi, R. et al. Focal adhesion kinase determines
the fate of death or survival of cells in response to
TNFα in the presence of actinomycin D. Biochim.
Biophys. Acta 1770, 518–526 (2007).
140. Leist, M. et al. Inhibition of mitochondrial ATP
generation by nitric oxide switches apoptosis to
necrosis. Exp. Cell Res. 249, 396–403 (1999).
141. Leist, M., Single, B., Castoldi, A. F., Kuhnle, S. &
Nicotera, P. Intracellular adenosine triphosphate (ATP)
concentration: a switch in the decision between
apoptosis and necrosis. J. Exp. Med. 185,
1481–1486 (1997).
This paper introduces the idea that intrinsic
differences in cellular energy levels might have a
key role in the selection of a cell’s form of death:
either apoptotic or necrotic.
142. Volbracht, C., Leist, M. & Nicotera, P. ATP controls
neuronal apoptosis triggered by microtubule
breakdown or potassium deprivation. Mol. Med. 5,
477–489 (1999).
143. Nicotera, P., Leist, M., Fava, E., Berliocchi, L. &
Volbracht, C. Energy requirement for caspase
activation and neuronal cell death. Brain Pathol. 10,
276–282 (2000).
144. Dimmeler, S., Haendeler, J., Sause, A. & Zeiher, A. M.
Nitric oxide inhibits APO‑1/Fas-mediated cell death.
Cell Growth Differ. 9, 415–422 (1998).
Acknowledgements
J.Y. is supported in part by grants from the National Institute
on Aging (NIA) Merit Award (R37 AG12859) and the National
Institutes of Health (NIH) Director’s Pioneer Award. A.D. is
supported in part by NIA Mentored Research Scientist Career
Development Award and Massachusetts Medical Foundation
Smith Family New Investigator Award.
DATABASES
Interpro: http://www.ebi.ac.uk/interpro
LRRs | NACHT | Pyrin
UniProtKB: http://beta.uniprot.org/
AIF | ANT | APAF1 | ASC | ATG5 | ATG7 | BAK | BAX | BCL2 |
BCL-XL | Beclin-1 | BID | BIM | Caspase‑9 | CED‑3 | CED‑4 |
CED‑9 | CIITA | CXCR4 | DAP | EGL‑1 | HMGB1 | MAPK8 |
MCL1 | NAIP | NALP1 | NLRC4 | NLRX1 | PARP1 | PIDD | RIP1 |
TNFα | TRAF2 |
FURTHER INFORMATION
Junying Yuan’s homepage:
http://www.hms.harvard.edu/dms/bbs/fac/yuan.html
Alexei Degterev’s homepage: http://www.tufts.edu/med/
biochemistry/faculty/degterev/degterev.html
All links are active in the online pdf