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Apoptosis:
Molecular Mechanisms
Min Wu, Harvard Medical School, Boston, Massachusetts, USA
Han-Fei Ding, Harvard Medical School, Boston, Massachusetts, USA
David E Fisher, Harvard Medical School, Boston, Massachusetts, USA
Secondary article
Article Contents
. Introduction
. Features of Apoptosis
. Apoptosis in Tissue Homeostasis, Disease and the
Immune System
. Inducers of Apoptosis and Apoptotic Signalling
. Fas and TNFa Receptor-mediated Apoptotic Signalling
Apoptosis is an intrinsic cell-suicide programme which ensures proper development by
maintaining tissue homeostasis and safeguarding the organism through the elimination of
unwanted or virus-infected cells.
Introduction
Apoptosis, derived from the Greek word for a natural
process of leaves falling from trees or petals from flowers, is
a distinct form of programmed cell death. Although such
programmed deaths were described many decades ago, the
significance of apoptosis had been largely overlooked,
particularly its relevance to disease. The improved understanding of apoptotic signalling pathways, and the cloning
and characterization of pro- or antiapoptotic genes have
attracted great interest to this process and raised the
possibility that therapeutic strategies which alter apoptotic
pathways may be useful in the treatment of cancer,
infectious diseases, degenerative syndromes and other
pathological conditions.
Features of Apoptosis
Cell death may occur via at least two broadly defined
mechanisms: necrosis or apoptosis. Necrosis is a passive,
catabolic, pathological cell death process which generally
occurs in response to external toxic factors such as
inflammation, ischaemic or toxic injury. It is not thought
to ever occur under physiological conditions. Necrosis is
characterized by the swelling of mitochondria, early
rupture of the plasma membrane, dispersed chromatin
and early destruction of the intact structure of the cell. In
contrast, apoptosis is an active, metabolic, genetically
encoded and evolutionarily selected death pathway. It
occurs under either physiological or pathological conditions. This suicidal pathway is characterized by membrane
blebbing, the appearance of highly condensed chromatin
and activation of an endonucleolytic process, which leads
to the sequential cleavage of chromosomal DNA to a size
of several hundred kilobases, then to 50 kb and eventually
to 200 bp. These oligosomal DNA fragments result in a
distinct laddering pattern on an ethidium bromide-stained
agarose gel that represents a hallmark of apoptosis (Wyllie
et al., 1980). As a result, cells shrink and condense into
. Genes that Regulate Apoptosis
. Executioners of Apoptosis: Caspases
. Summary
multiple small membrane-bound ‘apoptotic bodies’ which
display a particular propensity as targets for phagocytes.
This removes apoptotic cells without leaking the cytoplasmic contents into the intercellular space, minimizing tissue
inflammation, avoiding damage to neighbouring cells, and
efficiently degrading host (or viral) DNA.
Apoptosis in Tissue Homeostasis,
Disease and the Immune System
Apoptosis is a fundamental biological process that is
implicated in early development such as during metamorphosis in insects and amphibians, and organogenesis in
virtually all multicellular organisms. As examples of this
cellular altruism, apoptosis plays an active part in the
removal of interdigital webs in fingers and toes and in the
formation of T and B cell repertoires of the immune system
by eliminating nonreactive or self-reactive cells. This
programmed cell death is so precisely executed that in the
development of the nematode Caenorhabditis elegans,
exactly 131 cells die according to a well-regulated genetic
programme (Hengartner and Horvitz, 1994).
Apoptosis also exerts a role opposite to mitosis in the
maintenance of cell populations. As many as 1011 cells die
in an adult human per day to ensure tissue homeostasis,
and it is estimated that within a typical year, the mass of
cells a person loses through cell death is almost equivalent
to their entire body weight. Such death therefore probably
plays an important part in dynamic processes such as tissue
remodelling and responses to stress. Apoptosis is also a
protective mechanism, directing lysis of virus-infected
cells, foreign cells or incipient neoplasm. Excess cell death
can contribute to the acquired immune deficiency syndrome (AIDS) and neurodegenerative diseases like Alzheimer and Parkinson syndromes, and ischaemic injury
such as myocardial infarction. Too little cell death could
lead to cancer, persistent viral infection, or autoimmune
disorders.
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Apoptosis: Molecular Mechanisms
Apoptosis plays a central role in the immune system.
Under physiological conditions, immature lymphocytes
that bind to autoantigens are eliminated by apoptosis. This
is thought to protect against immune recognition of ‘self’.
Defects in the deletion of these lymphocytes predispose to
autoimmunity. Apoptosis in thymocytes is associated with
the elimination of self-reactive clones of developing T cells
following their interaction with antigens in the thymus. Fas
and Fas ligand, cell surface molecules belonging to the
tumour necrosis factor (TNF) family, are implicated in
downregulation of the immune reaction as well as T cellmediated cytotoxicity. Malfunction of the Fas system
contributes to lymphoproliferative disorders and autoimmune disease.
Apoptosis is a significant physiological mechanism for
establishing B-cell tolerance and shaping the B-cell
repertoire. B cells are subject to death by apoptosis
throughout most stages of their maturation and 60–70%
cell loss has been calculated during the pre-B to B cell
transition in bone marrow. Germinal centres are active
sites of normal B lymphocyte differentiation; hypermutation and proliferation of activated B cells occurs in these
microenvironments following antigen immunization. It
has been suggested that these B lymphocytes are eliminated
within the germinal centres, and recent evidence indicates
that soluble antigen causes enhanced apoptosis of germinal
centre B cells. Activated B cells prepared from human
germinal centres spontaneously undergo apoptosis when
placed in tissue culture, but this can be prevented by
stimulating the cells with immobilized anti-immunoglobulin, especially in combination with anti-CD40 antibody.
Inducers of Apoptosis and
Apoptotic Signalling
Numerous environmental factors can activate cell suicide,
and other factors can specifically antagonize apoptosis.
Activators of apoptosis include tumour necrosis factor a
(TNFa), Fas ligand (FasL), transforming growth factor b
(TGFb), Bax (and other proapoptotic Bcl-2 family
members), and glucocorticoids. In addition, aberrant
oncogene expression (e.g. c-myc), or normal tumour
suppressor gene function (such as p53) may trigger
apoptosis under specific conditions. In many cases,
simultaneous conflicting signals for growth stimulation
and suppression trigger apoptosis. Most growth factors
exert explicitly antiapoptotic signalling on their target
cells. Cytokines regulate survival through their receptors,
which trigger a cascade of intracellular signalling. Dozens
of cytokines function to promote cell survival, growth or
differentiation. In fact, recent studies have demonstrated
that loss of certain cell types due to mutation of a critical
growth factor could be largely rescued by targeted overexpression of the ‘generic’ antiapoptotic factor Bcl-2 in
2
growth factor-deprived cells. Results such as this suggest
that suppression of apoptosis is a major function of growth
factors, and differentiation pathways may represent the
default for precursor cells which survive. In this sense then,
growth factors would have less to do with specifying
lineage-specific differentiation per se. Among the intracellular (noncytokine) factors which have been shown to
potently suppress apoptosis are CD40 ligand, viral genes
such as E1B from adenovirus, baculovirus p35, and
antiapoptotic genes in 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/
propagation). A few apoptotic modulators, including
FasL and TNF, induce apoptosis which is largely confined
to the development and regulation of the immune system.
Fas and TNFa Receptor-mediated
Apoptotic Signalling
Fas receptor (CD95) belongs to a family of receptors
including the tumour necrosis factor (TNF) and nerve
growth factor (NGF) receptors that utilize related signalling pathways to regulate cell proliferation, differentiation
or death. CD95 (Fas/Apo-1), which plays a critical role in
T-cell mediated toxicity, and CD120a, a p55 TNF
receptor, are among the best studied examples (Nagata
and Goldstein, 1995; Salvesen and Dixit, 1997). Fas serves
as a receptor on the cell surface for a ligand (FasL), and the
crosslinking of FasL to Fas receptor triggers apoptosis on
the target cells. Fas is abundantly expressed in activated
mature lymphocytes, and in lymphocytes transformed
with human immunodeficiency virus (HIV) or human T
cell leukaemia virus (HTLV-I); in addition, certain tumour
cells also express Fas. The Fas apoptotic pathway is
implicated in eliminating unwanted activated lymphocytes
or virus-infected cells and its signalling pathway is
summarized in Figure 1.
Genes that Regulate Apoptosis
A large number of genes and proteins have been implicated
in the control of apoptosis. These can be categorized by
their activities at discrete steps in the apoptotic pathway as
well as their relationships to specific disease states (Figure 2).
A diverse assortment of triggers activate the cascade, which
is subject to tight homeostatic regulation by a number of
regulators or modulators of the death pathway. The ‘point
of no return’ in apoptosis is reached when caspases become
enzymatically active in cleaving target proteins (the
‘executioners’ of apoptosis). The Bcl-2 family of factors
regulate caspase activation either negatively (e.g. Bcl-2
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Apoptosis: Molecular Mechanisms
Receptors: Fas (Apo-1, CD95)
Ligands: Fas-L
DD domain
DED domain
Cleavage of Bid (mitochondria)
FADD adaptor protein
Procaspase-8
(MASH/FLICE)
Active caspase-8
Cleavage of procaspases
Cytochrome C release
Apaf-1, procaspase-9
Apoptosis
Figure 1 Fas receptor signalling pathways employ a cytoplasmic protein motif known as the death domain (DD) in the receptors and certain adaptor
proteins. The death domain is a conserved sequence of 80 amino acids. This DD motif is also found in adaptor proteins MORT1/FADD, TRADD and
RIP. The death domain of Fas binds to MORT1/FADD. In addition, MORT1/FADD interacts with caspase-8, a member of the ICE/Ced-3 protease family,
through another motif designated the death effector domain (DED). Recent evidence suggests that caspase-8 in turn cleaves Bid, a Bcl-2 family protein
which may regulate mitochondrial integrity in a manner which further activates the apoptotic cascade.
Triggers
Regulators
Executioners
damage
•
p53
•
•
cytokine starvation
•
death domain factors
• caspases
•
hypoxia
• Bcl-2
•
detachment
•
Myc/oncogenes
•
temperature
•
cytokine-responsive kinases
•
death receptor
•
cytochrome c
• DNA
Apaf-1
family
Figure 2 The apoptosis cascade: triggers, regulators and effectors (executioners). A variety of triggers both pathological and physiological (e.g. during
normal development) can activate apoptosis. Numerous regulators include factors which can dampen or amplify the apoptotic signal, as well as
intermediates which are essential participants in a specific apoptotic pathway (e.g. p53). Executioners are activated as downstream effectors. Their
activation represents a point of no return in the life or death of a cell.
itself) or positively (e.g. Bax). Other apoptosis modulators
reside further upstream and are thought to activate
cascades which are in turn subject to regulation by
downstream factors such as Bcl-2. Among these upstream
modulators are oncogenes such as c-myc which (seemingly
paradoxically) activates apoptosis in a manner which may
be important in tumorigenesis or cancer therapy. The
tumour suppressor p53 induces apoptosis under certain
conditions, thereby accounting for at least a portion of its
tumour suppressive activity. While regulation of apoptosis
is of paramount importance in cancer biology, an understanding of the relationship between cancer and apoptosis
has shed equally important light on other more general
biological phenomena, such as apoptosis in development.
Role of c-myc oncogene in control of apoptosis
c-myc, a proto-oncogene whose dysregulated expression
promotes cell proliferation and neoplastic transformation,
is aberrantly regulated in a large number of human
tumours and has been clearly implicated in control of
tumour cell apoptosis. The full-length human c-Myc
protein has 439 amino acids, is localized to the nucleus,
and has a short half-life. c-Myc is a transcription factor
which recognizes the CA[C/T]GTG element (E box), and
also has the ability to suppress transcription through a
pyrimidine-rich cis element termed the initiator (Inr).
Overexpression or inappropriate time of expression of cmyc has been found to promote apoptosis. It was observed
that ectopic expression of c-Myc protein accelerated
apoptosis following interleukin 3 (IL-3) deprivation of
the 32D IL-3-dependent myeloid cell line. Similarly Rat-1
fibroblast cells transfected with a Myc-oestrogen receptor
fusion protein (Myc-ER) expression vector led to enhanced levels of apoptosis upon growth arrest either by
serum deprivation or thymidine block (Evan et al., 1992).
Further, addition of antisense Myc oligonucleotides to
immature T cells and some T-cell hybridomas inhibited c-
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Apoptosis: Molecular Mechanisms
myc expression and prevented T-cell receptor-mediated
apoptosis. Together, these results suggest that inappropriate overexpression of c-myc promotes apoptosis. Myctriggered apoptosis has been particularly observed when
external conflicting signals (such as growth arrest) were
simultaneously imposed. In the absence of such conflicting
signals, overexpression of c-myc did not induce apoptosis
but rather caused cell cycle re-entry. Other studies have
suggested that the apoptotic pathway of c-Myc may
require wild-type p53, with Myc-induced apoptosis being
preceded by stabilization of p53. As described below, p53
appears to be an extremely important regulator of tumour
cell apoptosis, not necessarily restricted to tumours
harbouring Myc lesions.
The precise mechanism through which Myc activates
apoptosis remains enigmatic, although involvement of
certain factors such as the cell cycle phosphatase CDC25
has been suggested to play a role. Adding more confusion
to this pathway are reports that Myc-dependent apoptosis
is, in some contexts, independent of functional p53. 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 apoptosis of B cells.
These observations all eagerly await mechanistic explanations, but still highlight the fascinating paradox that
cancer, a disease traditionally viewed as excess proliferation and survival is inextricably linked to regulation of cell
death. Among the molecular regulators of greatest
importance in cancer cell apoptosis, p53 probably tops
the list.
Tumour suppressor gene p53 and apoptosis
The p53 protein was originally identified as a nuclear
phosphoprotein bound to the large transforming antigen
of the SV40 DNA virus (T antigen). It is now known to play
important roles in at least two major processes: regulation
of cell cycle progression and apoptosis.
Because p53 protein inhibits or reduces transformation
by viral and cellular oncogenes, p53 is classified as a
tumour suppressor gene. Furthermore, p53 mutations or
deletions comprise the most frequent genetic abnormalities
found in human tumours. 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 lose the ability to bind DNA and accordingly
fail to transactivate. Mixed 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 often act in a dominant
negative manner. Consistent with this notion, some
mutant forms of p53 neoplastically transform cells,
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presumably by inhibiting endogenous wild-type p53
function.
One of the important target genes transcriptionally
activated by p53 is the cyclin-dependent kinase (CDK)
inhibitor p21Cip/Waf1. The p53 protein elicits an increase in
p21 levels upon cellular damage inflicted by irradiation or
other external toxic agents, leading to CDK inhibition and
cell cycle arrest. The ability to arrest following DNA
damage permits repair, leading to the concept of p53 as a
watchdog for genomic fidelity. Extensive research is
focusing on the pathways that connect stresses such as
DNA damage to p53 activation. These include a variety of
kinases, including the product of the ataxia telangiectasia
disease gene (ATM) as well as the MDM2 oncoprotein
which modulates p53 degradation. MDM2 is in turn
regulated by interactions with a protein encoded in an
alternative reading frame at the p16/INK4a locus.
Although p53 modulates cell cycle arrest in response to
DNA damage, the p53 gene is largely dispensable for cell
growth or differentiation. The majority of p53-null mice
experience apparently normal embryogenesis, but develop
tumours with 100% penetrance within the first months of
life. Humans with hemizygous germline p53 aberrations
(Li-Fraumeni syndrome) also exhibit a high incidence of
early age malignancies.
The p53 protein has also been found to play a critical role
in apoptosis. This was first illustrated using a temperaturesensitive mutant of p53 which, when expressed in a myeloid
leukaemia cell line triggered massive apoptosis at the
permissive temperature (Yonish-Rouach et al., 1991).
Furthermore, thymocytes null for p53 function, due to
mutation or knockout, are profoundly resistant to radiation-induced apoptosis. Loss of p53 has also been shown to
confer significant resistance to apoptosis triggered by
many chemotherapeutic agents in genetically defined cell
lines. Correspondingly, clinical data in humans have linked
p53 loss to worsened prognosis in a large assortment of
tumour types. A very important, but incompletely understood, process is the mechanism by which p53 produces
apoptosis (versus arrest). A number of transcriptional
target genes have been identified, which are capable of
triggering death, although it is unclear what happens to
these genes when p53 produces arrest rather than death.
Separate studies have provided evidence that the apoptotic
activity of p53 may not induce transcription of new genes,
but employs a distinct pathway to activate caspases.
An important feature of p53 is the ability to induce
alternatively cell cycle arrest or apoptosis (Figure 3). In cell
culture studies, largely based on fibroblasts, p53 appears to
trigger arrest in primary cells, but apoptosis in oncogenetransformed cells. The mechanistic basis for this remains
unclear since a direct connection between p53 and caspases
has not yet been made. However a striking feature of this
regulation is that the context-dependent induction of
apoptosis by p53 may provide a therapeutic index which
‘translates’ DNA damage into an apoptotic signal in
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Apoptosis: Molecular Mechanisms
arrest
(untransformed cell)
Stress
p53
therapeutic
index
apoptosis
(oncogene transformed cell)
Figure 3 p53 regulates two alternative responses to stress: cell cycle arrest
or apoptosis. Diverse stresses, including DNA damage, many
chemotherapeutic drugs, growth factor deprivation, and others, induce
stabilization of p53 protein via signalling pathways which are incompletely
understood. In untransformed fibroblasts, this elevated p53 is associated
with transcriptional activation of genes which arrest the cell cycle. In
oncogene-transformed fibroblasts the elevated p53 is associated with an
apoptotic response (via unclear mechanisms). The differential arrest versus
apoptosis response endows p53 with the ability to produce cancer cellspecific death, a feature that correlates with its prognostic significance in
many human cancers.
cancer cells, but into an arrest signal in normal cells. Within
a cancer patient, one can then imagine that cancer
therapies may inflict identical damage throughout the
body, but p53 triggers apoptosis only in the cancer cells,
permitting cure (Fisher, 1994). Indeed, at present essentially all chemotherapy-curable tumours are among the
minority in which p53 remains wild type. While major
questions remain regarding the mechanisms underlying the
apoptotic activity of p53, it is likely that a better understanding of this pathway may lead to improved therapeutic
options for refractory (usually p53-deficient) cancers.
Role of NF-jB in receptor-mediated apoptosis
Nuclear factor-kB (NF-kB) was originally discovered as a
protein that bound specifically to an enhancer element (kB
site) of the immunoglobulin kappa light chain gene. Major
members of the NF-kB protein family include RelA (p65),
c-Rel and p50, and they have been implicated in the
regulation of a variety of important genes in the immune
response (e.g. Igk light chain, IL-2 and IL-2a), in
inflammatory and acute-phase response (e.g. interleukin
1(IL-1), IL-6, TNFa, and serum amyloid A protein), and in
certain viruses (e.g. HIV-LTR, SV40, cytomegalovirus and
adenovirus).
An antiapoptotic role for NF-kB/Rel was suggested by
Baltimore and coworkers. RelA (p65) knockout mice
exhibited embryonic lethality highlighted by massive liver
cell apoptosis, suggesting an antiapoptotic role for RelA.
In addition, it was known that TNFa led to NF-kB
activation, but was also associated with increased apoptosis. In this case, it appears that apoptosis occurs despite
NF-kB activation, which if anything blunts the apoptotic
response. NF-kB has been shown to protect cells from
apoptosis in a number of scenarios, including TNFa
signalling and anti-immunoglobulin M (anti-IgM)- or
TGFb-mediated apoptosis. The magnitude of killing
by TNFa is greatly enhanced when a dominant mutant
NF-kB inhibitor, IkB-a, is introduced into cells, preventing activation of endogenous NF-kB; furthermore, introduction of wild-type NF-kB impedes TNFa-induced
apoptosis.
These findings suggest strategies for enhancing anticancer treatment. Since the efficacy of an anticancer agent
would be reduced if it activates NF-kB, it would be
attractive to adopt combinations that inhibit NF-kB in the
presence of an apoptosis trigger. Inhibitors of NF-kB
activation, such as glucocorticoids, antioxidants and
naturally or synthetically derived inhibitors of NF-kB
might therefore be useful in combination with TNFa to
elicit a more effective killing of cancer cells. Still of
paramount importance is the requirement in cancer
therapy that enhanced apoptosis be selectively elicited in
cancer cells, but not normal cells. How the NF-kB pathway
will fit into such targeted killing remains to be determined.
Genes in control of apoptosis in the
nematode C. elegans
Major insights into the molecular mechanisms underlying
apoptosis have come from studies of the nematode C.
elegans, largely through the work of Horvitz and
colleagues. Due to the exquisitely detailed mapping of all
cell divisions and death events during development, it was
possible to identify mutants which globally influence these
apoptotic death events. Three major genes were identified:
two pro-apoptotic factors called Ced-3 and Ced-4 and an
antiapoptotic gene called Ced-9 (Hengartner and Horvitz,
1994). The product of Ced-3, the first known caspase, was
found to be homologous to human interleukin 1bconverting enzyme (ICE), a cysteine protease that processes IL-1b to the mature form during an inflammatory
response. Although ICE itself may have little to do with
apoptosis in mammals, additional cysteine protease
homologues are major effectors of apoptosis. The model
of C. elegans has also been valuable in identifying Ced-9, an
antiapoptotic gene, as a homologue of the Bcl-2 family.
Ced-4, a required effector of apoptosis, has been more
difficult to functionally dissect until the recent identification of a human homologue which resides in a caspase
regulatory complex (Figure 4).
Antiapoptotic genes
A variety of factors have been demonstrated to antagonize
apoptotic pathways, both during physiological events
(such as normal development) and in pathological states
(such as viral infections and cancer). Heavily studied
antiapoptotic genes include Bcl-2 and Bcl-XL in mammalian cells, Ced-9 in C. elegans, p35 in baculovirus, and E1B
19K protein in adenovirus.
Bcl-2 was first identified as part of a common translocation in human follicular lymphoma. A novel category of
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Apoptosis: Molecular Mechanisms
Apoptotic signals
Ced-4
Ced-3 (Caspase)
Cell death
Ced-9 (Bcl-2 family)
Positive regulator
Negative regulator
Figure 4 Major apoptosis regulators in C. elegans.
oncogene, Bcl-2 displayed the unusual property of extending cell survival rather than promoting cell proliferation
per se. Three conserved regions termed Bcl-2-homology
regions (BH1, BH2 and BH3) are required for protein–
protein interactions of importance in the regulation of
apoptosis. Bcl-2 is capable of dimerizing with a number of
related factors which, together with factors related by BH
motifs, comprise a family of apoptotic regulators (Chao
and Korsmeyer, 1998). Although their mechanisms of
action are not well understood, some Bcl-2 family members
actually promote apoptosis while others (like Bcl-2 itself)
antagonize apoptosis.
Bcl-2 expression is ubiquitous during embryogenesis,
but becomes more restricted in adult tissues associated
with long-term survival, such as stem cells and proliferating zones. Bcl-2 knockout mice are surprisingly normal at
birth, but with severely impaired lymphoid and kidney
development as well as age-related melanocyte loss. In
contrast, Bcl-2 overexpression targeted to the lymphoid
system extends B-cell survival with the persistence of
immunoglobulin secretion and frequent B-cell lymphoma.
Overexpression of Bcl-2 is capable of antagonizing
apoptosis triggered by c-myc or p53. However, Bcl-2
rescues apoptosis without affecting the mitogenic function
of c-Myc, nor reversing the growth arrest by p53,
indicating that Bcl-2 acts downstream of c-Myc or p53.
Bcl-X is a member of the Bcl-2 family. The Bcl-X gene
closely resembles Bcl-2, and functions to regulate cell
death. Bcl-X transcripts are alternatively spliced into two
products: Bcl-XL, the long (L) form and Bcl-LS, the short
(S) one. Bcl-XL has an open reading frame of 233 amino
acids and has domains similar to those of Bcl-2; Bcl-XS
encodes a protein of 170 amino acids with deletion of the
Bcl-2 homology domains BH1 and BH2. Bcl-XL acts to
inhibit apoptosis similarly to Bcl-2, whereas Bcl-XS
antagonizes the actions of Bcl-2 and Bcl-XL and is
proapoptotic. Bcl-XL antagonizes apoptosis in a variety
of tissues. Bcl-XL knockout mice die near embryonic day
13, confirming the important role of this antiapoptotic
gene in development. The three-dimensional X-ray crystal
structure of Bcl-X resembles that of the pore-forming
domain of bacterial toxins. Recent study shows that Bcl-X
can be embedded into either synthetic lipid vesicles or
planar lipid bilayers, and constitutes a pH-sensitive and
6
cation-selective ion-conducting channel at physiological
pH. It is therefore suggested that Bcl-X may sustain cell
survival by regulating the permeability of intracellular
membranes, or by maintaining mitochondrial membrane
potential.
Bax is another proapoptotic member of the Bcl-2 family.
It and other proapoptotic Bcl-2 family members may act as
heterodimer partners of Bcl-2 or Bcl-XL, and counteract
their ability to protect cell death. Several of the proapoptotic Bcl-2 family members have been connected with other
upstream regulators of the apoptosis cascade. Bax expression has been shown to be transcriptionally upregulated by
p53 in certain contexts. Another proapoptotic Bcl-2 family
member, Bad, is directly phosphorylated by the phosphatidylinositol 3-kinase target Akt, a potentially important
regulator of survival signals emanating from the cell
membrane. Bid, a third proapoptotic Bcl-2 family member,
was recently found to be a direct target of the Fas signalling
pathway, linking this extracellular death trigger to regulation of mitochondrial events and caspase activity.
Executioners of Apoptosis: Caspases
While there might be some philosophical discussion of
when death actually occurs in a cell triggered for apoptosis,
there is little disagreement that activation of caspases
represents a point of irreversibility. Caspases (cysteinyl
aspartate-specific proteinases) are a family of proteases
containing cysteine at their active sites. They are related to
mammalian interleukin 1b-converting enzyme (ICE/caspase-1) and to the nematode apoptotic gene product Ced-3
(Salvesen and Dixit, 1997). Since the discovery of the first
caspase in 1993, at least 10 different, related caspases have
been identified in humans. All these enzymes cleave
substrates just C-terminal to aspartic acid, and they are
grouped into three subclasses based on further features of
their substrate specificity. That these proteases play critical
roles in executing programmed cell death is exemplified by
caspase-3 knockout mice, most of which die in utero, and
the remaining of which survive with overall brain masses
twice the normal volume, apparently due to the existence of
supernumerary and ectopic cell masses that would have
undergone apoptosis during normal development. Sur-
ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net
Apoptosis: Molecular Mechanisms
prisingly, these defects are largely confined to the brain,
suggesting that caspase-3 dominates in regulating neuronal
apoptosis. A similar situation was seen in caspase-9
knockout mice. These caspase-9 deficient mice also died
with enlarged and deformed cerebra due to insufficient
apoptosis during brain development (Yoshida et al., 1998).
As expected, caspase-1 deficient mice cannot produce
mature IL-1b, but exhibit no other major development
abnormality, suggesting that it plays little if any role in
developmentally important apoptotic pathways. A large
number of caspase substrates have been identified, including the enzyme poly ADP ribose polymerase (often used as
a marker for caspase activity). It is unclear whether any
individual substrate cleavage represents the true death
blow, or whether it is the sum of all cleavages which
collectively dooms the cell.
Much progress has been made in recent years in the
analysis and development of caspase inhibitors (Nicholson
and Thornberry, 1997). Uncleavable peptide pseudosubstrates serve as potent experimental reagents and lead
compounds for medicinal chemistry. The inhibition of
caspases is also one of the major strategies adopted by
viruses to elude their destruction through suicide of the
infected cell (with associated endonucleolytic DNA
degradation). Cytokine response modifier A (CrmA) is a
product of cowpox virus that inhibits apoptosis of host
cells and functions as a potent inhibitor for caspase-1 and
caspase-8 (MACH, FLICE), but a weak inhibitor of
caspase-3. P35, another protein from baculovirus, also
attenuates apoptosis through inhibition of caspases. P35
inhibits caspases with looser specificity, having the ability
to abolish activity of almost all caspases. However, no
counterparts of P35 protein have been found to date in
mammals. Endogenous mammalian inhibitors of caspases
are the IAP family (inhibitors of apoptosis), polypeptides
that potently inhibit caspase-3 and caspase-7 and attenuate
apoptosis in a number of species.
One of the most difficult, but important mechanistic
questions in the regulation of apoptosis involves an
understanding of the precise pathway(s) leading to caspase
activation. Caspases exist in living cells as zymogens,
proenzyme polypeptides, which undergo proteolytic activation. They can in turn autoactivate other procaspase
molecules, potentially amplifying the effector wing of the
apoptotic cascade once the initial caspase activation has
occurred. It has been difficult to determine how this
activation comes about.
One relatively simple shortcut pathway is employed by
cytotoxic T cells, whose product, granzyme B, directly
cleaves procaspase molecules, thereby activating the
suicide enzymes in cells targeted for destruction. Another
mechanism has recently been elucidated by Wang and
colleagues (Zou et al., 1997). Using cell-free extracts, it was
shown that deoxy-ATP (dATP) and cytochrome c could
cooperate with caspase-9 and a mammalian Ced-4 homologue (termed Apaf-1) to activate caspases. In turn it was
shown that cytochrome c is released from mitochondria in
cells destined to undergo apoptosis, but prior to measurable activation of caspases. Thus leakage of cytochrome c
from mitochondria might serve as a key signal to activate
procaspases and initiate the biochemical events of death.
Apaf1 knockout mice revealed decreased apoptosis in the
brain and exhibited hyperproliferation of Apaf-1 plays a
crucial role in the mitochondrial pathways of apoptosis
(Yoshida et al., 1998). Since many Bcl-2 family members
localize to mitochondria, it is possible that regulation
upstream of caspases may occur at the level of maintaining
mitochondrial integrity (at least integrity to cytochrome c
leakage).
Summary
Apoptosis involves a cascade of complex events which
include the delivery of external signals through defined
receptor complexes, the well-regulated expression of a
number of genes, and the execution of apoptosis by
proteases and endonucleases. There may be crosstalk
between proapoptotic and antiapoptotic components
within the cells, and while other biochemical events may
be measured in terms of degrees of activation, apoptosis
represents a more cut-and-dry switch: it is either on or off.
Our understanding of this pathway has shed light on a
unique example of biological altruism, death of the
individuals for the sake of the whole, in a manner
particular to multicellular organisms. With a still better
understanding of the molecular regulators of this death
pathway it is anticipated that enormous opportunities will
arise to devise targeted therapies both for diseases of excess
cell death and diseases of insufficient cell death.
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Apoptosis: Molecular Mechanisms
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ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net