Apoptosis: Molecular

Mechanisms
Fatima Cairrao, ITQB, Oeiras, Portugal
Pedro M Domingos, ITQB, Oeiras, Portugal

Advanced article
Article Contents
. Introduction
. Features of Apoptosis
. Executioners of Apoptosis: Caspases
. Conclusions

Based in part on the previous version of this Encyclopedia of Life Sciences

(ELS) article, Apoptosis: Molecular Mechanisms by Min Wu, Han-Fei
Ding and David E Fisher.

Apoptosis is an intrinsic cell-suicide programme, which

ensures tissue homeostasis and safeguards the organism
by eliminating unnecessary and unwanted cells, or cells
that may constitute some form of danger to the organism,
for example, tumour cells. Both during embryonic development and in the adult life of organisms, there is a need
for a perfect balance between cell proliferation, differentiation and death. This balance is achieved, in part,
through the precise regulation of apoptosis, which
involves complex molecular events that ultimately activate, or prevent the activation of caspases (cysteineaspartic acid proteases). The activation of caspases is, in
most cases, irreversible and represents the commitment
to the cell death fate.

Introduction
Apoptosis, derived from the Greek word for the natural
process of leaves falling from trees, is a distinct form of
programmed cell death (Kerr et al., 1972; Kerr, 2002).
Although such programmed deaths were described many
decades ago, the signicance of apoptosis was largely
overlooked, in particular, its relevance to disease. During
the past 25 years, the improved understanding of apoptotic
signalling pathways, with the cloning and characterization
of pro- or antiapoptotic genes, has attracted great interest
to this process and raised the possibility that therapeutic
strategies altering apoptotic pathways may be useful for the
treatment of cancer, infectious diseases, degenerative syndromes and other pathological conditions.

ELS subject area: Developmental Biology

How to cite:
Cairrao, Fatima; and Domingos, Pedro M (January 2010) Apoptosis:
Molecular Mechanisms. In: Encyclopedia of Life Sciences (ELS). John
Wiley & Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0001150.pub2

Online posting date: 15th January 2010

Features of Apoptosis
Cell death may occur via at least two broadly dened
mechanisms: necrosis or apoptosis. Necrosis is a cell death
process generally occurring in response to trauma generated
by external factors or overwhelming cellular injury. Necrosis
is characterized by swelling and rupture of the plasma cell
membrane (cell lyse), with the release of the cellular contents
into the immediate extracellular space, which may cause
inammation or harm other neighbouring cells.
In contrast to necrosis (a dirty form of cell death),
apoptosis is a genetically encoded and evolutionarily conserved form of cell death, in which any harm done to the
organism by this process is minimized (a clean form of cell
death). Apoptosis is characterized by several morphological and biochemical aspects: the condensation of the
nucleus and cytoplasm, the activation of caspases and
nucleases (which degrade cellular proteins and deoxyribonucleic acid (DNA), respectively), membrane blebbing
and the fragmentation of cells into multiple small
membrane-bound apoptotic bodies, which are rapidly
phagocytosed by neighbouring cells. As a result, apoptotic
cells are removed from tissues without leaking their cytoplasmic contents into the intercellular space, minimizing
tissue inammation and avoiding damage to neighbouring
cells. Apoptosis may occur during normal physiological
conditions, for example, during embryonic development,
where unnecessary cells may die by apoptosis and deregulation of apoptosis may cause pathological conditions such
as cancer or neurodegenerative diseases (Jacobson et al.,
1997; Vaux and Korsmeyer, 1999). See also: Apoptosis:
Morphological Criteria and Other Assays; Apoptosis:
Regulatory Genes and Disease

Executioners of Apoptosis: Caspases

Although there might be some discussion of when death
actually occurs in a cell triggered for apoptosis, there is little
disagreement that, in most cases, activation of caspases
represents a point of irreversibility in the cell death process.
The pioneering work of H Robert Horvitz and colleagues
with the nematode Caenorhabditis elegans established that
the apoptotic gene ced-3 is a caspase, belonging to a family of
proteases containing cysteine at their active site and being

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Apoptosis: Molecular Mechanisms

related to the mammalian interleukin 1b-converting enzyme

(Yuan et al., 1993). Since the discovery of the rst caspase,
many more members of this family have been identied in
humans and in other species. All these enzymes are constitutively expressed in virtually all cells as inactive zymogens
(procaspases) and, on activation, cleave substrates just
C-terminal to aspartic acid residues. The activation of caspases is itself dependent on the proteolytic cleavage of the
zymogens, which leads to the removal of an inhibitory Nterminal domain and the production of two subunits (one
large and one small). These subunits associate as a heterotetramer (two large and two small) to form the active protease that cleaves many cellular proteins. Among the targets
of caspases are other molecules of caspase zymogen,
potentially amplifying the apoptotic cascade once the initial
caspase activation has occurred. Therefore, much eort has
been made in addressing the important mechanistic questions in the regulation of caspase activation and understanding the precise pathway(s) leading to the activation of
apoptosis. See also: Apoptosis: Morphological Criteria and
Other Assays; Interleukins.

Regulation of caspase activation

The regulation of caspases occurs by two distinct molecular
signalling pathways, depending on whether the cell signals

activating apoptosis originate extracellularly, thereby

activating the extrinsic pathway (more on this in the sections ahead), or intracellularly, thereby activating the
intrinsic pathway (Figure 1).

Intrinsic pathway
Most of the current knowledge about the molecular
mechanisms regulating the intrinsic pathway derives from
studies using the nematode C. elegans or mammalian systems (Figure 1). In the classic pathway, the activation of
caspases results from the formation of a multiprotein
complex, termed the apoptosome, consisting of CED-4/
Apaf-1, procaspase-9 and cytochrome c (in mammals
only), which is required for the initial activation of procaspase-9, with the activation of other caspases occurring
subsequent to caspase-9 activation (Shi, 2006). For this
reason, caspase-9 homologues are also known as initiator
caspases, whereas caspases that are directly or indirectly
activated downstream of caspase-9 to participate in the
degradation of the cellular components, such as caspase-3,
are known as eector caspases. See also: The Apoptosome:
The Executioner of Mitochondria-mediated Apoptosis
Members of the B-cell lymphoma protein 2 (Bcl-2)
family of proteins are important regulators of caspase-9
activation, since they can facilitate or prevent the release of

Debcl/Buffy

EGL-1

Bcl-2

Bax

CED-9

CED-4

CED-3

Apaf-1

Ark/Dark

Reaper
Hid
Grim

Diap1

Dronc

Smac/Diablo
HtrA2/Omi
Arts

IAPs

Drice
Dcp-1

Caspase9

Effector
caspases

Figure 1 The intrinsic pathway in C. elegans, D. melanogaster and mammalians. The functional homologues are represented in boxes with the same colour.
The activation of effector caspases occurs downstream of the activation of initiator caspases (CED-3, Dronc and caspase-9), which occurs on the formation of
the apoptosome, a protein complex contaning CED-4, Ark/Dark and Apaf-1, and cytochrome c. In mammalians, the release of cytochrome c from
mitochondria is a critical step for caspase activation, which is regulated by members for the Bcl-2 family of proteins. In Drosophila, the importance of
cytochrome c and Bcl-2 family members (Debcl/Buffy) for apoptosome formation and caspase activation is still not clear. In C. elegans, CED-4 directly
activates CED-3 with no apparent requirement of cytochrome c for this process to occur. The negative regulation of CED-4 by CED-9 is blocked by EGL-1, a
BH3-only member of the Bcl-2 family of proteins. IAPs (in mammalians) or Diap1 (in Drosophila) directly bind to caspases via their BIR (baculovirus inhibitory
repeat) domains, to inhibit caspase activity. In Drosophila, reaper, grim and hid are three Diap1 antagonists, which directly bind Diap1 via the short Nterminal peptide motif termed IBM (IAP-binding motif) present in all three proteins, to release caspases from the negative interaction with Diap1. In
mammalians, Smac/Diablo, HtrA2/Omi and ARTS (apoptosis-related protein in the TGF-signalling pathway) can function as antagonists of IAPs.

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Apoptosis: Molecular Mechanisms

cytochrome c from mitochondria into the cytoplasm and

regulate the formation of the apoptosome (Danial and
Korsmeyer, 2004). A second branch of the intrinsic pathway is an inhibitory one, where caspase activation
is blocked by the inhibitor of apoptosis proteins (IAPs) and
is only unleashed on expression of IAP antagonists, such as
reaper, grim and head involution defective (hid) in the
fruity, Drosophila melanogaster, or Smac/Diablo in
mammalians (Song and Steller, 1999). It appears that both
these pathways are used in coordination to control the
activation of caspases, in a manner similar to way the gas
and the brake are used in the process of driving an automobile (Steller, 2008), but the contribution of each branch
may vary according to the cell type and signalling paradigm. See also: Inhibitor of Apoptosis (IAP) and BIRcontaining Proteins

Extrinsic pathway
The decision to undergo apoptosis may also be determined
by the balance between proapoptotic and antiapoptotic
signalling events triggered by environmental (extracellular)
factors, such as Fas ligand (FasL/CD95L), tumour necrosis
factor a (TNFa), transforming growth factor b (TGFb) and
cytokines. Most growth factors and cytokines promote cell
survival, growth and dierentiation, by triggering antiapoptotic signalling on their target cells. In fact, the loss of
certain cell types due to mutations of critical growth factors
can be rescued by targeted overexpression of the generic
antiapoptotic factor Bcl-2. Results such as this suggest that,
in certain situations, suppression of apoptosis is a major
function of growth factors and cytokines, and that cell
dierentiation may represent the default pathway for precursor cells that survive. Among the intracellular (noncytokine) factors that have been shown to potently suppress
apoptosis are the CD40 ligand, viral genes such as E1B from
adenovirus or p35 from baculovirus and antiapoptotic
members of the Bcl-2 family. A large number of DNA
viruses have been demonstrated to encode factors which
function to curtail the cellular apoptotic response, presumably a prerequisite for successful viral infection and
propagation. See also: Cytokines; Death Receptors; Oncogenes; Transforming Growth Factor Beta: Role in Cell
Growth and Dierentiation
In mammals, the extrinsic pathway mediates apoptosis
in response to the activation of cell-surface death receptors,
such as Fas/CD95 and TNFa receptor. Death receptors
can induce apoptosis directly, through the activation of
caspases or indirectly, by amplifying the death signal
through the activation of the intrinsic/mitochondrial
pathway. All Fas receptors contain a conserved extracellular death receptor domain (DR), where FasL binds,
inducing the oligomerization of death receptors and initiating a cascade of events that leads to the activation of
apoptosis in the target cells. Activated death receptors bind
the adaptor molecule Fas-associated death domain
(FADD) via the death domain (DD) and FADD recruits
the initiator procaspase-8 and procaspase-10 into a

complex, the death-inducing signalling complex (DISC),

through the death eector domain (DED), which is present
both in FADD and in the procaspase. The recruitment of
procaspase-8 and procaspase-10 into the DISC complex
leads to the autoproteolytic cleavage and activation of
these caspases, with subsequent activation of the eector
caspases (Figure 2). See also: Mitochondrial Outer Membrane Permeabilization
Fas plays a critical role in T-cell mediated toxicity, being
abundantly expressed in activated mature lymphocytes
and in lymphocytes transformed with human immunodeciency virus (HIV) or human T-cell leukaemia virus
(HTLV-I). It is believed that the Fas apoptotic pathway is
implicated in eliminating unwanted activated lymphocytes
or virus-infected cells.
Contrary to the evolutionary conserved intrinsic pathway, the components of the extrinsic pathway are not
conserved in all metazoan organisms. In C.elegans, no celldeath receptors of the TNF family have been found so far.
In D. melanogaster, only a single TNF ligand (Eiger) and its
associated receptor (Wengen) are present and it is believed
that Eiger/Wengen activate the c-Jun N-terminal kinase
(JNK) pathway via the adaptor proteins DTRAF1/2,
inducing apoptosis indirectly. The Drosophila homologues
of caspase-8 (Dredd) and FADD do not seem to be
involved in the activation of apoptosis, but are instead
required for the production of antimicrobial peptides in
response to Gram negative (2) bacteria (Kuranaga and
Miura, 2007).
Disregulation of the Fas-mediated apoptosis can lead to
several pathological disorders, with reduced Fas-mediated
apoptosis being associated with excessive cell proliferation
in hepatocellular carcinoma and excessive Fas-mediated
apoptosis being associated with viral and alcoholic hepatitis, Wilson disease and hepatic brosis (Guicciardi and
Gores, 2009).

Bcl-2 family members

The members of the Bcl-2 family of proteins are important
components of the intrinsic pathway, regulating mitochondrial outer membrane permeabilization (MOMP) and
the release of proapoptotic factors, such as cytochrome c,
from mitochondria. In addition, Bcl-2 family members are
key regulators of apoptosis because they connect the
extrinsic and intrinsic pathways (Figure 2). Bcl-2 was rst
identied as being the cause of human follicular lymphoma
due to a chromosome translocation aecting Bcl-2 function. Subsequently, it was shown to promote tumuorigenesis by inhibiting apoptosis, instead of promoting cell
proliferation (Chao and Korsmeyer, 1998).
In humans, there are presently 23 known members of the
Bcl-2 family (Hardwick and Youle, 2009), all of which
contain at least 1 of the 4 possible protein domains of
homology (BH Bcl-2 homology). Bcl-2 family members
can be classied into three categories according to their
functions, as well as the number and type of BH protein

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Apoptosis: Molecular Mechanisms

Ligand (FasL)
Extrinsic
pathway

Mitochondrial
pathway

Death receptor
(FAS)

Smac/
Diablo

DED

DED

DD

DISC
complex

DED

DD
DD

Bcl-xL

HtrA2/
Omi

Bax
Bak

Adaptor
(FAD)

MOMP
tBid

Procaspace
(Caspase-8/-10)

Cyt C
Release

Bid

IAPs

Caspase-8
Caspase-10
Apaf-1

Caspase-9

Apoptosome

Activation of
effector caspases
(caspase-3, -6, -7)

Figure 2 The extrinsic pathway mediated by the FAS death receptor. FasL activates the Fas receptor by binding to the extracellular death receptor domain. Fas
receptor also contains a cytoplasmic motif known as the death domain (DD), which is also found in the adaptor proteins FADD, TRADD and RIP. The DD of Fas
binds to the DD of FADD, whereas FADD interacts with procaspase-8/-10, through another motif designated death effector domain (DED). The formation of
this complex of FAS/FADD/procaspase-8/-10 (DISC complex) is required for the activation of caspases. Caspase-8, in turn, cleaves both effector caspases and
Bid, a proapoptotic member of the Bcl-2 family of proteins. The processed Bid (tBid) activates Bax and Bak, members of the Bcl-2 family that oligomerize to
promote MOMP. tBid inhibits the function of the antiapoptotic members of the Bcl-2 family (Bcl-2 and Bcl-xL), which normally prevent the oligomerization of
Bax and Bak, inhibiting MOMP and apoptosis. MOMP allows the release of various proapoptotic mitochondrial proteins such as cytochrome c, Smac/Diablo and
HtrA2/Omi that further activate the apoptotic cascade. Cytochrome c induces the heptamerization of the cytosolic protein Apaf-1, which binds procaspase-9 to
form the active apoptosome complex for cleavage of effector caspases, whereas SMAC and HtrA2 act as inhibitors of IAPs.

domains: antiapoptotic proteins with multiple BH domains (BH1BH4), proapoptotic proteins with multiple
domains (BH1BH3) and proapoptotic proteins with
BH3-only domain (Table 1).
In the prevailing model for regulation of apoptosis by Bcl2 family members (Figure 2), the antiapoptotic multidomain
members, such as Bcl-2, bind to and neutralize the proapoptotic members (Bax) in nonapoptotic cells. On receiving
death-inducing signals, BH3-only domain proteins inactivate the antiapoptotic multidomain proteins, releasing the
proapoptotic proteins from the inhibitory interaction with
the antiapoptotic proteins. The proapoptotic Bcl-2 family
proteins then oligomerize, creating pores in the mitochondrial outer membrane and allowing the release of cytochrome c into the cytoplasm, which leads to caspase
4

Table 1 Classication of Bcl-2 family members according to

their apoptotic role and the type and number of BH domains
Function in
apoptosis

BH domains

Member proteins

Antiapoptotic
Proapoptotic
Proapoptotic

Multiple
Multiple
BH3-only

Bcl-2, Bcl-xL, Mcl-1

Bax, Bak, Bok
Bid, Bad, Bim, Puma

activation and cell death. In an alternative model, the antiapoptotic members directly bind and inhibit the BH3-only
domain proteins, which otherwise directly induce the oligomerization of the proapoptotic multidomain proteins.

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Apoptosis: Molecular Mechanisms

Recently, much more attention has been paid to the

additional, nonapoptotic roles of Bcl-2 family proteins,
including those in biological processes like the regulation of
mitochondrial dynamics (fusion and ssion of mitochondria) or autophagy (the process in which cells undergoing nutrient starvation degrade cellular components
to survive and maintain a minimal metabolic activity).
See also: Autophagy in Nonmammalian Systems; Mitochondria Fusion and Fission
In C. elegans, there are three Bcl-2-related proteins:
CED-9, EGL-1 and CED-13. CED-9 is an antiapoptotic
multidomain protein, which inhibits CED-4 (Apaf-1)mediated activation of CED-3 (caspase). During apoptosis, CED-9 is inhibited by the BH3-only proteins EGL-1
and CED-13 to allow the activation of CED-3 (Figure 1).
Two Bcl-2 family proteins exist in D. melanogaster, Buy
and Debcl, both containing multiple BH domains. However, Bcl-2 family members seem to have a limited role in
the regulation of apoptosis in Drosophila, whereas the Diap
pathway seems more preponderant in this biological
system (Steller, 2008). See also: The Bcl-2 Family Proteins Key Regulators and Eectors of Apoptosis

Role of the c-myc oncogene in the control of

apoptosis
Additional factors have been shown to directly or indirectly control apoptosis, including the proto-oncogene
c-myc, identied 25 years ago, which has a central role in
the regulation of growth control, cell dierentiation and
apoptosis, and is among the genes that most frequently
contribute to the development of human tumours. c-MYC
is a transcription factor which recognizes the CA[C/
T]GTG element (E box) and also has the ability to repress
transcription through a pyrimidine-rich cis element termed
the initiator (Inr). The target genes of c-MYC are many,
being involved in a variety of physiological processes, such
as cell cycle regulation, metabolism, protein synthesis and
cell adhesion. See also: Transcriptional Gene Regulation in
Eukaryotes
Overexpression or inappropriate expression in time of
c-MYC has been found to promote apoptosis. In the early
1990s, it was observed that ectopic expression of c-MYC
protein accelerated apoptosis in cells deprived of survival
factors (Homan and Liebermann, 2008). Still, other
observations have suggested that in B cells MYC may
regulate apoptosis in precisely the opposite fashion:
inhibition of c-MYC resulted in dramatic apoptosis,
whereas overexpression of c-MYC protected B cells from
apoptosis. Further, addition of antisense c-MYC oligonucleotides to immature T cells and to some T-cell hybridomas inhibited c-MYC expression and prevented T-cell
receptor-mediated apoptosis. The precise molecular
mechanisms of how MYC induces apoptosis remain
unclear. However, it appears that multiple pathways are
regulated by MYC, including one requiring the p53 tumour
suppressor, where MYC-induced apoptosis is preceded by
stabilization of p53. In mammalian cells, p53 is a major

regulator of cell cycle arrest and apoptosis (see following

sections). Deregulated MYC upregulates ARF (acute renal
failure), which in turn activates p53 to regulate a group of
target genes that activate apoptosis and cell cycle arrest
(Figure 3). The loss of ARF and p53 caused an increase in
tumourigenesis in mouse models, conrming the importance of the ARFMDM2 (mouse double minute 2)p53
pathway in MYC-induced apoptosis. Other reports have
suggested, however, that MYC-dependent apoptosis is,
in some contexts, independent of functional p53. This
dependence on p53 seems to be determined by the cell
type and the apoptotic signal triggered (Homan and
Liebermann, 2008).
In Drosophila, only one Myc protein (dMyc) has been
identied, with similar biochemical and molecular characteristics to its mammalian orthologue. dMyc is also
involved in the regulation of cell proliferation and apoptosis, and the functional conservation between the Drosophila and the mammalian orthologues has been shown by
complementation studies in both organisms. However,
dMyc diers from its vertebrate orthologue by the fact that
its overexpression does not lead to the formation of
tumours. Also, the apoptotic pathway induced in Drosophila is dierent from the mammalian one. Despite the
existence of a p53 orthologue in Drosophila, neither ARF
nor MDM2 orthologues are present in the y genome.
Recently, it was shown that the apoptosis caused by dMyc
in Drosophila imaginal disc cells occurs by the process of
cell competition: in neighbouring cells expressing dierent
levels of dMyc, either due to dMyc loss-of-function
mutations or overexpression of dMyc, the cells with higher
levels of dMyc out-compete the cells with lower levels of
dMyc, which undergo apoptosis (Moreno and Basler,
2004; de la Cova et al., 2004). One explanation for this
phenomenon is that cells with higher levels of dMyc proliferate faster and sequester growth factors, which normally are produced in limited amounts, causing apoptosis
in cells with lower levels of dMyc due to the lack of growth
factors. Other studies have shown that dMyc induced
apoptosis is accompanied by the induction of Drosophila
p53 messenger ribonucleic acid (mRNA), but that dp53
activity is not essential for dMycs ability to induce
apoptosis.

Tumour suppressor gene p53 and apoptosis

The p53 protein was originally identied as a nuclear
phosphoprotein that binds the large transforming antigen
of the SV40 DNA virus (T antigen). Since then, p53dependent signalling pathways have been the subjects of
intense study (Fuster et al., 2007) (Vousden and Prives,
2009). p53 is now recognized to act as a transcription factor
regulating the expression of genes involved in at least two
major processes in response to DNA damage or cellular
stress: regulation of cell cycle progression and apoptosis.
The rst evidence of the involvement of p53 in apoptosis
was obtained by introducing a temperature-sensitive
mutant of p53 in a myeloid leukaemia cell line. At the

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Apoptosis: Molecular Mechanisms

Hypoxia
DNA damage
Oncogenic activation
Ribosomal stress

Cytoplasmic
(e.g.MYC)

Nuclear

ARF

Apoptosis:
P

p p53

p53

MDM2

p p53

Senescence:

Activity
stabilization
Ub

- p21

p53

Bcl-xL

26S
proteosome
p53

Bax
Bak

Ub

Cell cycle arrest:

(G1 phase)
DNA repair

p53

Bcl-xL
Puma

Ub

- PAI-1

Puma

Ub

- PUMA
- NOXA
- MDM2
- Apaf-1
- Bax
- Fas

p53

MOMP
Mitochondrial outer membrane
Figure 3 Regulation of apoptosis by p53. Several stress conditions (DNA damage, hypoxia, oncogene activation, among others) lead to the transcriptional
activation and an increase of the p53 protein levels. Activation of p53 promotes the transcriptional activation of many target genes involved in several cellular
responses: apoptosis, cell cycle arrest, DNA repair and senescence. Under normal physiological conditions, the cytoplasmic p53 protein is maintained at low
levels by MDM2 (an E3 ubiquitin ligase) that targets the p53 protein to ubiquitylation (Ub) and subsequent degradation by the proteosome. MDM2 is also a
target of the nuclear p53 protein and MDM2 activity is counteracted by ARF in response to activation of oncogenes, such as c-MYC. Inhibition of MDM2
allows p53 to accumulate, both in the nucleus and the cytoplasm. In the cytoplasm, p53 can interact with proteins of the Bcl-2 family, being sequestered by
Bcl-xL at the mitochondrial outer membrane. PUMA, which is a transcriptional target of nuclear p53, is capable of disrupting the Bcl-xL/p53 interaction,
allowing p53 to interact with Bax/Bak proteins and inducing MOMP and the subsequent cascade of the mitochondrial apoptotic pathway (Green and
Kroemer, 2009).

permissive temperature, the p53 mutant triggered massive

apoptosis of the cells. In genetically dened cell lines, loss of
p53, due to mutation or knockout, confers resistance to
apoptosis triggered by radiation and by chemotherapeutic
agents. In broblasts, p53 triggers cell cycle arrest in primary cells, but apoptosis in oncogene-transformed cells. In
humans, the loss of p53 comprise the most frequent genetic
abnormality found in human tumours and is associated to
the worst prognosis. Up to 50% of human cancers contain
deleted or mutated p53 genes, including 80% of colon
cancers, 50% of lung cancers and 40% of breast cancers.
Many human p53 mutants have been described; most
mutants loose the ability to bind DNA and accordingly fail
to activate the transcription of target genes. Complexes of
wild-type and mutant p53 protein are unable to bind the
p53 site or transcriptionally activate p53 reporter constructs, suggesting that mutant p53 proteins may act in a
dominant negative manner. See also: Chronic Lymphocytic Leukaemia; Epstein-Barr Virus; Tumour Suppressor
Genes
The kind of cellular response following p53 activation
is dependent on the type of cell and nature of the cellular stress. Recently, a novel transcription-independent
6

proapoptotic function, mediated by the cytoplasmic form

of p53, revealed that p53 can participate directly in the
regulation of the apoptotic intrinsic pathway, by interacting with members of the Bcl-2 family to induce mitochondrial outer membrane permeabilization (Green and
Kroemer, 2009).
One of the important target genes transcriptionally
activated by p53 is the cyclin-dependent kinase (CDK)
inhibitor p21. The p53 protein elicits an increase in p21
levels on cellular damage inicted by radiation or other
external toxic agents, leading to CDK inhibition and cell
cycle arrest. The arrest of the cell cycle following DNA
damage allows time for DNA repair to occur, leading to the
concept that p53 acts as a guardian for genomic delity.
Induction of p21 expression is very sensitive to even low
levels of p53. This ne regulation permits a transient arrest
in the G1 phase of the cell cycle under mild DNA damage
or stress and the survival of cells until optimal cellular
conditions are restored. However, in the case of precancerous lesions, tumour progression can be avoided by
an irreversible cell cycle arrest: p53 mediated senescence.
The p53 tumour suppressor pathway acts therefore as a
surveillance mechanism that is activated in response to

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Apoptosis: Molecular Mechanisms

cellular stress. Despite its involvement in cell cycle arrest,

the p53 gene is largely dispensable for cell growth or
dierentiation.
Modulation of mammalian p53 activation is regulated
at the levels of transcription and translation, as well at
the posttranslational level (Lavin and Gueven, 2006).
Activation of p53 can occur in consequence of several
posttranslational modications, such as phosphorilation,
ubiquitination, acetilation and other proteinprotein
interactions, that can either stabilize p53 protein or convert
p53 into its active phosphorilated form, changing its subcellular localization (nuclear export and/or mitochondrial
association). Modulators of p53 include MDM4, that
inhibits p53 transcriptional activity, a variety of kinases,
such as ATM (product of the ataxia telangiectasia disease
gene) and the ubiquitin ligase MDM2. MDM2 stimulates
nuclear export of p53 by monoubiquilation, leading to p53
proteasomal degradation in the cytoplasm. On DNA
damage sensor proteins (RAD proteins, HUS1) induce
ATM/ATR which phosphorylate the eector kinases
(CHK1/CHK2), which modulate phosphorylation of p53.
In unstressed cells, p53 protein is maintained at low levels
due to its interaction with the E3 ubiquitin ligase MDM2.
Dierent cellular stresses can lead to the stabilization of the
p53 protein, and an important mechanism for p53 protein
stabilization is via the ARF-dependent inactivation of
MDM2 (Figure 3).
In C. elegans, the p53-like protein (CEP-1) only shares
homology with the DNA-binding domain of its mammalian homologue, and is required for the radiation-induced
apoptosis in the germ cells. CEP-1 mediates several stressinduced responses but not a cell cycle arrest upon DNA
damage. CEP-1 is required for the induction of EGL-1 and
CED-13 (proapoptotic BH3-only proteins) after DNA
damage. In Drosophila p53 (dmp53) is required for transcriptional induction of the proapoptotic proteins Reaper,
Hid and Sikcle. Activation of dmp53 induced by Drosophila CHK2 phosphorylation, but both in C. elegans and
Drosophila regulation of p53 homologues by MDM2 is
absent (Lu and Abrams, 2006).

The role of NFjB in apoptosis

The nuclear factor-k-light-chain-enhancer of activated B
cells (NFkB) is a central mediator of immune and inammatory responses in mammals. NFkB originally discovered
as a protein that bound specically to an enhancer element
of the immunoglobulin kappa light chain gene in activated
B cells. Major members of the NFkB protein family include RelA (p65), Rel B, c-Rel, NFkB1(p105/p50), and
NFkkB2(p100/p52), which have been implicated in the
regulation of a variety of important genes in the immune
response (IL-2 and IL-2a), in inammatory and acute-phase
response (interleukin 1 (IL-1), IL-6, TNFa, and serum
amyloid A protein) and in response to certain viruses (e.g.
HIV-LTR, SV40, cytomegalovirus and adenovirus).
In resting cells, cytosolic NFkB dimers are bound to IkB
inhibitory proteins that prevent the nuclear translocation

of NFkB and the transcriptional activation of NFkB target genes. Two distinct NFkB activation cascades, that
respond to dierent stimuli, can be triggered: the canonical
(or classical) NFkB pathway and the noncanonical (or
alternative) NFkB signalling cascade (Dutta et al., 2006).
The NFkB canonical pathway is activated in response to
injury, inammation, infection and other stress conditions.
Some of the target genes activated by the NFkB pathway
include antiapoptotic factors of the Bcl-2 family (Bcl-xL,
Bcl-2). The antiapoptotic role of NFkB factors was rst
demonstrated by the embryonic lethality of RelA-decient
mice, which develop massive liver cell apoptosis. Transcriptional activity of NFkB factors is inhibited by IkB
proteins that mask the DNA-binding ability of NFkB
dimers (p60/p50) by sequestering them in the cytoplasm.
This inhibitory activity of IkB is counteracted by IkB
kinases (IKK), that promote phosphorylation and degradation of IkB by the proteosome and release of NFkB
dimers.
NFkB has been shown to protect cells from apoptosis in
a number of scenarios, including TNFa signalling and
antiimmunoglobulin M (anti-IgM)- or TGFb-mediated
apoptosis. Apoptosis induced by TNFa signalling can be
greatly enhanced when a dominant inhibitor of NFkB,
IkB-a, is introduced into cells, preventing activation of
endogenous NFkB; furthermore, introduction of wild-type
NFkB impedes TNFa-induced apoptosis. However under
specic circumstances proapoptotic activity of NFkB can
also occur. NFkB can induce the transcription of proapoptotic target genes, including the Fas death receptor and
respective death-inducing ligand, p53 and the proapoptotic
Bcl-2 family members Bax and Bcl-xL cascade (Dutta et al.,
2006).

Conclusions
Apoptosis involves a cascade of complex events, from
external apoptotic signals activating specic receptor
complexes to the execution of apoptosis by activation of
proteases and endonucleases. The commitment to apoptosis depends on the balance between proapoptotic and
antiapoptotic signalling components within cells. The
understanding of these signalling pathways and molecular
regulators opens enormous opportunities that may lead to
specic therapies for diseases caused by deregulation of the
normal cell death processes.

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ENCYCLOPEDIA OF LIFE SCIENCES & 2010, John Wiley & Sons, Ltd. www.els.net