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A POPTOSIS S IGNALING
Vishva Dixit, Andreas Strasser

Annual Review of Biochemistry

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 Annu. Rev. Biochem. 2000. 69:217–45
Copyright c 2000 by Annual Reviews. All rights reserved

APOPTOSIS SIGNALING
Andreas Strasser1, Liam O’Connor1, and Vishva M. Dixit2
1The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia;
e-mail: strasser@wehi.edu.au
2Genentech Incorporated, South San Francisco, California 94080;

e-mail: dixit@gene.com

Key Words cell death, Bcl-2 protein family, tumor necrosis factor receptor family,
cysteine proteases, development
■ Abstract Apoptosis, a physiological process for killing cells, is critical for the
normal development and function of multicellular organisms. Abnormalities in cell
death control can contribute to a variety of diseases, including cancer, autoimmunity,
and degenerative disorders. Signaling for apoptosis occurs through multiple indepen-
dent pathways that are initiated either from triggering events within the cell or from
outside the cell, for instance, by ligation of death receptors. All apoptosis signaling
pathways converge on a common machinery of cell destruction that is activated by a
family of cysteine proteases (caspases) that cleave proteins at aspartate residues. Dis-
mantling and removal of doomed cells is accomplished by proteolysis of vital cellular
constituents, DNA degradation, and phagocytosis by neighboring cells. This article
reviews current knowledge of apoptosis signaling, lists several pressing questions, and
presents a novel model to explain the biochemical and functional interactions between
components of the cell death regulatory machinery.

CONTENTS
BACKGROUND AND HISTORY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
MORPHOLOGICAL AND MOLECULAR CHANGES
DURING APOPTOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
THE MAJOR PLAYERS IN APOPTOSIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Caspases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Adaptor Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
The Tumor Necrosis Factor Receptor Family . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
The Bcl-2 Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
APOPTOSIS SIGNALING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
APOPTOSIS AND NORMAL PHYSIOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
APOPTOSIS AND DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
A MODEL FOR THE BIOCHEMICAL ACTION
OF THE BCL-2 FAMILY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
CONCLUSIONS AND PERSPECTIVE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

0066-4154/00/0707-0217/$14.00 217
218 STRASSER ¥ O’CONNOR ¥ DIXIT

BACKGROUND AND HISTORY

The number of cells in an organ is determined by the rates of cell immigration,

cell division, and cell death (1). The phenomenon of physiological cell death has
been discovered independently several times over the past 150 years (2–4). The
impact of this process on cell production and homeostasis was widely recognized,
however, only after the discovery that cell death in multicellular organisms is
subject to genetic control (5, 6) and after the demonstration that abnormalities
in cell death regulation can cause diseases such as cancer (7, 8), autoimmunity
(9, 10), and possibly degenerative disorders (11, 12). More recent highlights in
apoptosis research include the discovery of death receptors and their ligands (13–
15), definition of the separate apoptosis signaling pathways, and identification of
the components of the common cell death effector machinery and their biochemical
functions (14, 16–18).

MORPHOLOGICAL AND MOLECULAR CHANGES

DURING APOPTOSIS
Electron microscopic analysis has identified the morphological changes that oc-
cur during apoptosis, including chromatin condensation, cytoplasmic shrinkage,
and plasma membrane blebbing (3, 19). These studies have also noted that, dur-
ing the early stages of apoptosis, no visible changes occur in mitochondria, the
endoplasmic reticulum, or the Golgi apparatus. However, others have more re-
cently reported swelling of the outer mitochondrial membrane (20) and release
of cytochrome c (21, 22) and apoptosis-inducing factor, an oxidoreductase-related
flavoprotein (23), from the mitochondrial intermembrane space. Several compet-
ing models have been proposed to explain how these events could be involved in
apoptosis regulation, and these are discussed below.
Molecular changes induced during apoptosis include internucleosomal DNA
cleavage (24) and randomization of the distribution of phosphatidyl serine (PS) be-
tween the inner and outer leaflets of the plasma membrane (25). Techniques based
on detecting some of these changes, such as terminal deoxynucleotidyltransferase-
mediated incorporation of labeled nucleotides at DNA breaks (26), staining of cell
surface-exposed PS with annexin V (27), and detection of cells with apparent
subdiploid DNA content by staining with DNA intercalating dyes (28), are now
standard tools for demonstrating apoptosis.
These morphological and molecular changes are elicited by a broad range of
physiological or experimentally applied death stimuli and are observed in cells
from diverse tissue types and species. This is consistent with the idea that inde-
pendent signaling pathways converge on a common death effector machinery that
is evolutionarily conserved (29–31).
 APOPTOSIS SIGNALING 219

THE MAJOR PLAYERS IN APOPTOSIS

Recent advances have led to the identification of four major functional groups of
molecules involved in triggering and affecting the apoptotic process. These are the
caspases, the adaptor proteins, which control the activation of initiator caspases,
members of the tumor necrosis factor (TNF) receptor (TNF-R) super family, and
members of the Bcl-2 family of proteins.

Caspases
A group of cysteine proteases, now called caspases (17, 32), is essential for pro-
grammed cell death in a variety of species (33, 34). Cysteine protease activity
can be detected in all cells undergoing apoptosis, regardless of their origin or the
death stimulus. The Ced-3 protein encodes an aspartate-specific cysteine protease
(35, 36) that is essential for all somatic cell deaths that occur during normal devel-
opment in Caenorhabditis elegans (31). Mice lacking caspase-3 or caspase-9 have
increased numbers of neurons in the brain, and their lymphocytes are partially
resistant to some apoptotic stimuli (37–40). Moreover, transgenic expression of
baculovirus protein p35, a potent inhibitor of all known caspases (41), prevents de-
velopmentally programmed cell death in C. elegans and Drosophila melanogaster
and inhibits apoptosis in a variety of mammalian cell lines (42–45). It is therefore
beyond doubt that caspases are not merely responsible for degradation of cellular
substrates during the end stage of apoptosis, but are critical regulators of cell death
initiation.
At least 14 caspases have been identified in mammals (32–34; Figure 1). These
enzymes recognize tetrapeptide motifs and cleave their substrates on the carboxyl
side of an aspartate residue. Individual caspases have distinct substrate specificities
that are determined by the pattern of amino acids upstream of the cleavage site
(the P2–P4 positions; 34). Caspases are synthesized as zymogens, which have very
low intrinsic enzymatic activity. The fully active enzymes are heterotetramers
composed of two identical subunits of ∼20 kDa plus two identical subunits of
∼10 kDa (46–48). These subunits can be produced by caspase-mediated cleavage.
There is evidence that aggregation of at least some caspase zymogens is sufficient
to promote self-processing (49–51). So-called initiator caspases (e.g. caspase-8
and caspase-9) start an avalanche of increasing caspase activity by processing and
activating so-called effector caspases (34).
Some caspases, particularly effector caspases, cleave and inactivate certain vi-
tal cellular proteins, such as DNA repair enzymes, lamin, gelsolin, MDM2 (an
inhibitor of p53), and protein kinase Cδ (17, 34). There are also enzymes that
can be activated directly or indirectly by caspase-mediated proteolysis. Certain
caspases (e.g. caspase-3) can remove a negative regulatory domain from the ki-
nase p21-activated protein kinase 2, and this is thought to trigger plasma mem-
brane blebbing (52). The caspase- activated DNase (CAD) is normally inactivated
by binding to an inhibitor, iCAD [also called DNA fragmentation factor (DFF)
220 STRASSER ¥ O’CONNOR ¥ DIXIT

Figure 1 Structural comparison of the caspase pro-enzymes and substrate specificity of the
caspases. The aspartate cleavage sites between the large (open bars) and small (tinted bars)
subunits are indicated. **, Aspartate cleavage site is not known. Death effector domains (DED)
and caspase recruitment domains (CARD) are shown. Human caspases 1–10, mouse caspases
11–14, and C. elegans Ced-3 are shown.

(53–55)]. During apoptosis, iCAD is cleaved by caspases, and this leads to release
of the active endonuclease, which produces the characteristic internucleosomal
DNA cleavage.

Adaptor Proteins
Adaptor proteins are the links between the cell death effectors, caspases, and the
cell death regulators, death receptors, and Bcl-2 family members. These links
take the form of physical associations between members of the three classes of
molecules, with the adaptor proteins forming bridges between caspases and up-
stream regulators of apoptosis. Associations between adaptor proteins and cas-
pases or TNF-R family members are characteristically mediated by homotypic
interactions between domains known as the death domain (DD), the death ef-
fector domain (DED), and the caspase recruitment domain [CARD (14, 56;
Figure 2)].
 APOPTOSIS SIGNALING 221

Figure 2 Adaptor proteins, showing death domains (DD), death effector domains (DED), and
caspase recruitment domains (CARD). Apaf-1 and Ced-4 have conserved ATPase domains, and
Apaf-1 also has WD40 repeats at its C terminus. FLIPL inhibits FADD mediated activation of
caspase-8.

The DD is a region in the cytoplasmic part of CD95 and related TNF-R family
members, as well as adaptor molecules such as Fas-associating death domain pro-
tein/mediator of receptor-induced toxicity (FADD/MORT1), TNF-R1-associated
death domain protein (TRADD), and receptor-interacting protein (RIP). After
cross-linking of a TNF-R family member containing a DD, homotypic interactions
between its DD and that of the adaptor allow caspase aggregation and activation
(57–60). Caspase recruitment and aggregation are themselves mediated by an-
other domain found in adaptor molecules, the DED. As well as a DD, FADD has
a DED. Tandem repeats of DEDs are also present in the pro-domains of caspase
8 and 10 zymogens. Cross-linking of CD95 can therefore result in pro-caspase-8
aggregation and activation through FADD.
Death receptor → FADD → caspase-8 signaling can be blocked by FLICE-
inhibitory protein (FLIP) molecules, which prevent recruitment and activation of
pro-caspase-8 (61–67). There are two splice forms of FLIP. The longer, more
abundant form, FLIPL, closely resembles pro-caspase-8 but lacks key residues
in the catalytic site and other regions that are thought to contribute to substrate
binding. The shorter form, FLIPS, consists only of the two DED domains.
Not all initiator caspases contain a DED, just as not all initiator caspases are acti-
vated through TNF-R cross-linking. Mammalian pro-caspase-9 and pro-caspase-2,
as well as C. elegans Ced-3, contain CARDs, which are also present in their specific
adaptors Apaf-1 and Ced-4. By a mechanism not as thoroughly characterized as
that of caspase-8 activation through CD95, apoptotic stimuli that are controlled by
pro-apoptotic members of the Bcl-2 family result in Apaf-1–mediated activation
of caspase-9 (16).
222 STRASSER ¥ O’CONNOR ¥ DIXIT

The Tumor Necrosis Factor Receptor Family

Members of the TNF-R family have pleiotropic action. Depending on the cell type
and the other signals that the cell receives, these receptors can trigger prolifera-
tion, survival, differentiation, or death (13–15). These receptors are activated by
a group of structurally related ligands that belong to the TNF ligand family (Fig-
ure 3). Most of these ligands are synthesized as membrane-anchored trimers, and
it appears that extensive receptor cross-linking is required for signaling (68–70).
CD95 (also called Fas or APO-1) and TNF-RI, as well as some other members of
this family, contain a cytoplasmic region, the DD, which is essential for inducing
apoptosis (71, 72). Members of this subfamily are sometimes referred to as death
receptors.
Much has also been learned about other death-inducing ligands and their re-
ceptors. TRAIL/APO-2L (TNF-related apoptosis-inducing ligand; TRAIL) in-
duces apoptosis preferentially in transformed cells, and in contrast to other death-
inducing ligands, it is expressed in a wide range of tissues (73). Four receptors for
TRAIL/APO-2L have been identified (designated DR4, DR5, DcR1, and DcR2 or
TRAIL-R1–TRAIL-R4), but only DR4 and DR5 can trigger apoptosis. DcR1 and
DcR2 lack the intracellular and transmembrane regions or the death domain, re-
spectively, and therefore cannot induce cell death (74–78). These receptors might
act as decoys and thereby inhibit TRAIL/APO-2L–mediated cytotoxicity. A decoy
receptor for FasL (DcR3) has also been found to be overexpressed in lung and colon
tumors (79). A single receptor for the APO-3 ligand, called DR3, APO-3, TNF
receptor-related apoptosis-mediating protein (TRAMP), WSL-1, or lymphocyte-
associated receptor of death (LARD), has been discovered (80–84). Experiments
with specific caspase inhibitors or embryonic fibroblasts from caspase-deficient
mice demonstrated that caspase-8 is essential for DR4-, DR5-, and DR3-transduced
apoptosis (74, 75, 77, 85, 86). It is controversial whether these receptors activate
caspase-8 through FADD and/or TRADD or whether they use presently unknown
adaptors (77, 87–90).
Members of the TNF-R family can also activate the transcriptional regulators
NF-κB and Jun kinase (JNK). Signaling through TRADD and the kinases RIP,
NF-κB–inducing kinase (NIK), IκB kinase (IKKα), and IKKβ culminates in phos-
phorylation and degradation of the IκB family of NF-κB inhibitors. Signals through
TRAF molecules activate JNK by a presently unknown mechanism (14, 15). Ex-
periments with mice that are deficient in RIP or TNF receptor-associated factor 2
(TRAF2) demonstrated that these signals promote cell survival rather than apop-
tosis, consistent with an anti-apoptotic role for the NF-κB and JNK pathways (91–
93). Surprisingly, FADD is not only needed for death receptor-induced apoptosis
but also plays an essential role in mitogen-induced proliferation of T lymphocytes
(94, 95). The nature of this cell growth signaling pathway is not known, but it is
predicted not to involve NF-κB and JNK (96).
 APOPTOSIS SIGNALING 223

Figure 3 Structural comparison of the members of the TNF receptor family. The extracellular
ligand-binding regions of the receptors are characterized by variable numbers of cysteine-rich
repeats. Death receptors contain a death domain in their intracellular region, which is essential for
apoptosis signaling. Decoy receptors DcR1 and DcR2 compete with DR4 and DR5 for binding
to TRAIL, and DcR3 is a soluble receptor for FasL. The members of the TNF receptor family
function as trimers and multimers of trimers, but for the sake of simplicity, single polypeptide
chains are shown. All proteins shown are mammalian, except CAR1, which is from the chicken.
224 STRASSER ¥ O’CONNOR ¥ DIXIT

Figure 4 Structural comparison of the members of the Bcl-2 protein family. The Bcl-2 family
of proteins can be divided into two subgroups, those that inhibit apoptosis and those that enhance
it. Numbered regions 1–4 indicate the BH1–4 Bcl-2 homology domains. Transmembrane (TM)
domains are indicated in gray. Only the C-terminal 200 amino acids of Mcl-1 are shown.

The Bcl-2 Family

A number of proteins with structural similarity to Bcl-2, the prototypical inhibitor
of apoptosis, have been discovered during the past 5 years (16). Mammalian Bcl-xL
(97), Bcl-w (98), A1/Bfl-1 (99, 100), Mcl-1 (101), and Boo/Diva (102, 103), C.
elegans Ced-9 (104), and a number of viral homologs also promote cell survival
and have three or four regions with extensive amino acid sequence similarity
with Bcl-2 ( Bcl-2 homology regions BH1–BH4). More distantly related family
members, including mammalian Bax (105), Bcl-xS [a splice variant of the bcl-x
gene (97)], Bak (106–108), and Bok/Mtd (109) contain 2 or 3 BH regions and
enhance apoptosis under conditions of stress. The most potent inducers of cell
death are a group of proteins that possess only a BH3 region and otherwise bear no
resemblance to the Bcl-2 family or any other presently known protein (Figure 4).
This group of pro-apoptotic molecules includes mammalian Bad (110), Bik/Nbk
(111, 112), Bid (113), Hrk/DP5 (114), Bim/Bod (115, 116), and Blk (117) and
C. elegans Egl-1 (118). Pro-apoptotic and anti-apoptotic members of the Bcl-2
protein family can physically interact (119), but it is presently unknown whether
this interaction occurs in the form of dimers or as part of a conglomerate of
molecules (see below).
 APOPTOSIS SIGNALING 225

Many members of the Bcl-2 family have a conserved C-terminal transmembrane

region. This region localizes proteins to the outer leaflet of the nuclear envelope,
the outer mitochondrial membrane, and the endoplasmic reticulum (120–126).
Subcellular localization studies by electron microscopy and biochemical fraction-
ation have established that the bulk of the Bcl-2 protein is on the cytosolic aspect
of these membranes. Similar subcellular localization has been reported for Bcl-xL,
A1, Mcl-1, Bax, and Bak (127–131). Bcl-2 family members naturally lacking a
predicted transmembrane region or mutant forms in which it is deleted can still
localize to these membranes, albeit less efficiently, indicating that the sites of
intrafamily interactions are at these membranes (132–134). This subcellular local-
ization appears to be fixed for some family members, but others can change their
subcellular localization. It has been reported that Bax is a cytosolic protein until
the cell is triggered to undergo apoptosis, when it becomes redistributed to mito-
chondrial membranes (135, 136). It is possible that such subcellular redistribution
may be a mechanism of regulating the activity of Bcl-2 family members.
Remarkably little overall sequence similarity is needed for similar function
among the pro-apoptotic Bcl-2 family members. Because all of the pro-apoptotic
proteins so far discovered have a BH3 region and not all of the anti-apoptotic
proteins have this region, this may be a defining characteristic for the two subfam-
ilies. For instance Bik/Nbk, Bid, Bim, and Hrk/DP5 have only the BH3 domain
in common, yet all are pro-apoptotic when overexpressed, and all bind to anti-
apoptotic Bcl-2 family members (111–114). The limited common features of the
pro-apoptotic members of the Bcl-2 family (all they share is a 16-amino-acid
BH3 domain) may mean that each has some specific activity, such as interaction
with specific upstream signaling molecules, and that their common function is to
bind and antagonize the anti-apoptotic effects of their ligands. This implies that
the biochemical effects of the Bcl-2 family on apoptosis are mediated through the
anti-apoptotic members. This has important implications when a model of how the
Bcl-2 family may regulate apoptosis is suggested, and these are discussed below.
Comparative studies in cell lines and in transgenic mice have shown that path-
ways to apoptosis that can be inhibited by Bcl-2 can also be inhibited by Bcl-xL,
Bcl-w, A1, and Mcl-1. Similarly, death stimuli that are insensitive to Bcl-2 are
also insensitive to its homologs (98, 99, 137–139). Most telling, a bcl-xL transgene
was able to substitute for bcl-2 in lymphocytes of Bcl-2–deficient mice (140). The
existence of several genes encoding proteins with similar biochemical action al-
lows the expression of anti-apoptotic regulators in a tissue- and stimulus-specific
manner. This idea is supported by the findings that Bcl-2 expression can be regu-
lated by cytokines that signal through receptors containing the γ c chain (141, 142);
A1 and Mcl-1 are induced by colony-stimulating factors (143, 144); and Bcl-xL
expression can be regulated by mitogens (145).
The activity of different pro-apoptotic Bcl-2 family members is controlled by
distinct post-translational modifications. The activity of Bad can be regulated by
Akt or protein kinase A-mediated phosphorylation (134, 146–148), Bid is acti-
vated through caspase-mediated proteolysis (149, 150), and Bim is regulated by
226 STRASSER ¥ O’CONNOR ¥ DIXIT

binding to the Dynein motor complex (151). It is therefore possible that these
molecules act as sentinels of cellular damage at distinct sites and that different
apoptotic stimuli induce cell death by activating distinct members of the Bcl-2
family. Post-translational modification is not limited to pro-apoptotic members of
the Bcl-2 family. Bcl-2 and Bcl-xL have been shown to be regulated by phospho-
rylation (152–154), and Bcl-xL and Ced-9 can be caspase substrates (155, 156).
The physiological relevance of post-translational modifications of anti-apoptotic
Bcl-2 family members is presently unclear.

APOPTOSIS SIGNALING

Considerable insight into the nature of the cell death effector machinery has been
derived from genetic studies in C. elegans and their comparison with results from
genetic and biochemical experiments in mammalian cells. All developmentally
programmed deaths of somatic cells in C. elegans require three proteins: the
caspase Ced-3, the adaptor protein Ced-4, and Egl-1, a BH3-only pro-apoptotic
member of the Bcl-2 family. The Bcl-2 homolog Ced-9 is needed for cell survival
(30, 31). Protein-protein interactions between Ced-3, Ced-4, Ced-9, and Egl-1 pro-
teins have made a direct link between the caspases, the effector arm of the cell
death pathway, and the Bcl-2 protein family. The connecting element between the
two types of proteins appears to be Ced-4, and its mammalian homolog, Apaf-
1, is thought to fulfill a similar function (157–162). Transgenic experiments in
C. elegans (163) and the discovery that binding of Ced-9 to Ced-4 is essential for
its survival function (158, 161, 164) implied that Ced-4 acts upstream of or in par-
allel to the Ced-3 caspase, and that Ced-9 acts as an inhibitor of Ced-4. Ced-4 can
bind simultaneously to the caspase Ced-3 and to Ced-9. This mechanism appears
to be conserved in mammals, because the human Ced-4 homolog Apaf-1 can bind
to and activate human caspase-9 zymogens, and this can be inhibited by Bcl-xL
(165–168).
Experiments with transgenic and gene knockout mice have shown that dif-
ferent initiator caspases, together with their specific adaptors and regulators, are
required for control and execution of different death stimuli (Figure 5). In thymo-
cytes and embryonic fibroblasts, caspase-9 and its adaptor, Apaf-1, are needed
for DNA damage-, corticosteroid-, and staurosporine-induced cell killing, but
they are dispensable for CD95 (Fas/APO-1)- and TNF-RI–transduced apopto-
sis (39, 40, 169, 170). In contrast, caspase-8 and its adaptor, FADD, are needed
for CD95- and TNF-RI–transduced apoptosis, but they are dispensable for the
other pathways to cell death (86, 94, 95, 171, 172). Bcl-2 and its homologs are po-
tent inhibitors of cell death caused by growth factor deprivation, DNA damage,
or treatment with corticosteroids or staurosporine, all of which require Apaf-1
and caspase-9. Conversely, in at least some cell types, particularly lymphocytes,
Bcl-2 and its homologs are poor antidotes to apoptosis transduced through CD95
or TNF-RI (137, 138, 173, 174). Consistent with these observations, Bcl-2 and its
 APOPTOSIS SIGNALING 227

Figure 5 Distinct pathways to apoptosis converge on activation of caspases. Apoptosis can be

induced by stimulation of those members of the TNF receptor family, which have a death domain
(e.g. CD95), or by a broad range of stimuli including growth factor withdrawal. These pathways
to effector caspase activation are distinct. Caspase-8–mediated activation of the pro-apoptotic
BH3-only protein Bid may provide a link between these pathways in some cell types.

homologs have been shown to interact with Apaf-1 and prevent it from activating
caspase-9 (167, 168). However, FADD-induced activation of caspase-8 and apop-
tosis are not blocked by these anti-apoptotic molecules (50, 96). It therefore appears
that mammals have two distinct mechanisms for activating effector caspases. One
is initiated by stress-induced signals inside the cell, requires Apaf-1 and caspase-9,
and is regulated by the Bcl-2 protein family. The other is activated by CD95 and
related receptors, requires FADD and caspase-8, and cannot be blocked by Bcl-2
or its homologs. It has been reported that Bcl-2 can inhibit hepatocyte apoptosis
caused by injection of antibodies to CD95 (175,176) and that Bcl-2–insensitive and
Bcl-2–inhibitable pathways leading from CD95 to apoptosis can coexist within the
single cell (177). However, all of these experiments were performed with antibod-
ies to CD95, and it is now known that their action is not identical to that of FasL
(178). It is therefore imperative to reevaluate CD95 signaling in experiments by
using the physiological ligand (70).
228 STRASSER ¥ O’CONNOR ¥ DIXIT

A possible connection between the pathways controlled by TNF-R family

members and those controlled by the Bcl-2 family has been identified. It has
been shown that caspase-8 can cleave Bid, a death-inducing member of the Bcl-2
family (149, 150). However, apoptosis triggered through CD95 was unaffected in
lymphoid cells from mice lacking Bid, although liver cells were refractory to anti-
CD95 antibody treatment (179). These findings suggest that, because proteolytic
Bid activation occurs downstream of caspase activation, the point of no return in
apoptosis control, it may not be essential for cell death in all tissues, but may be
required to amplify the caspase cascade in cells with low amounts of caspase-8.
It is likely that the CD95 → FADD → caspase-8 pathway is of physiological
significance. FADD and caspase-8 can be coimmunoprecipitated with activated
CD95 signaling (DISC) complexes (59, 60), and loss of FADD function or inhibi-
tion of caspase-8 rendered T lymphocytes and other cells completely resistant to
CD95-transduced apoptosis (94, 95, 137, 171, 172, 174). It is not yet clear whether
the three more speculative CD95 signaling pathways—CD95 → RIP → RAIDD
→caspase-2, CD95 → Daxx → ASK1 → JNK → unknown caspase, and CD95 →
FLASH → unknown caspase or adaptor—are of physiological importance. For in-
stance, Daxx was found to be localized exclusively in the nucleus (180, 181), and
mice lacking Daxx have enhanced, rather than suppressed, apoptosis (182).
Genetic and biochemical studies in mammals and C. elegans have indicated
that anti-apoptotic Bcl-2 family members keep cells alive by preventing Apaf-
1/Ced-4–like adaptor proteins from activating caspase zymogens. In C. elegans,
programmed cell death is abolished in mutants lacking the egl-1 gene, which
encodes a pro-apoptotic member of the Bcl-2 protein family with only a BH3
domain (118). It therefore appears that the cell death machinery is activated when
pro-apoptotic members of the Bcl-2 family bind to anti-apoptotic members and
thereby free Apaf-1/Ced-4–like adaptor proteins to activate caspase zymogens. It is
important to determine which transcriptional and post-translational mechanisms
activate pro-apoptotic Bcl-2 family members, what signaling mechanisms con-
verge on adaptor proteins, and what the exact composition of the death-inducing
protein complex apoptosome is.
Experiments with cell extracts have shown that holocytochrome c and dATP or
ATP promote Apaf-1–mediated activation of pro-caspase-9 (183, 184). In addition,
holocytochrome c has been found to migrate from mitochondria to the cytoplasm
in cells undergoing apoptosis, and this process was shown to be activated by pro-
apoptotic Bcl-2 family members and hindered by their anti-apoptotic relatives
(21, 22, 149, 150, 185). It is presently not clear whether this process is essential for
initiation of the caspase cascade or acts as an amplifier (186; see below).

APOPTOSIS AND NORMAL PHYSIOLOGY

The scientific literature is full of examples showing that a large number of chemical
or biological compounds can trigger apoptosis, in at least some cell types under
certain conditions. This has been interpreted as evidence that a given stimulus is
 APOPTOSIS SIGNALING 229

physiologically or clinically important, but this is often not the case. This sec-
tion concentrates on those apoptotic stimuli that are the most relevant to normal
physiology, development, and tissue homeostasis.
Cell death is critical for animal development. In mammals, some developmental
cell deaths are autonomous, meaning that pro-apoptotic signals from neighboring
cells are not needed for the demise of the doomed cell (187). In fact it is be-
lieved that, in many of these deaths, neighboring cells provide survival signals
and that cell death is initiated by withdrawal of growth factors and/or loss of
cell attachment (188). These pathways to cell death are often referred to as death
by neglect and can, in many instances, be blocked by anti-apoptotic members of
the Bcl-2 protein family (189). Consistent with this, mice lacking Bcl-2 or Bcl-
xL exhibited specific defects in organogenesis (190, 191). Other developmental
cell deaths are by design and require apoptosis-inducing signals from neighbor-
ing cells (187). Death receptors and their ligands are prime candidates for this
function. A role for the TNF-R family in development can be inferred from the
fact that defects in its signal transducers FADD or caspase-8 cause cardiac abnor-
malities and early embryonic lethality (86, 171). The absence of obvious devel-
opmental defects in those mice lacking members of the TNF or TNF-R families
so far studied is likely to be explained by functional overlap between related
molecules (14). As we have discussed above, these two physiologically activated
cell death pathways are subject to distinct control. Death by neglect is regulated
by the Bcl-2 protein family, and, in neuronal tissues, it requires Apaf-1, caspase-
9, and caspase-3 (37–40, 169, 170). Not all death by neglect requires caspase-9
and Apaf-1, however, because the spontaneous death of cultured thymocytes from
mice lacking Apaf-1 or caspase-9 appears normal (39, 169, 170). This may mean
that some other initiator caspase can be activated by Apaf-1 in caspase-9–null
cells, and it may also imply that mammals have Ced-4–like adaptors other than
Apaf-1.
Cell loss through apoptosis occurs in many tissues at multiple stages of cell
differentiation. Mice expressing bcl-2 transgenes have been used to determine
which of these physiological cell deaths occur by a mechanism that can be blocked
by Bcl-2. For example, Bcl-2 expression restored normal development and function
of T lymphocytes in mice lacking the interleukin-7 receptor (192, 193) but did not
block the death of thymocytes bearing autoreactive antigen receptors (194–196)
or those unable to express a pre–T-cell receptor (192, 197). This indicates that
two (or more) distinct pathways to physiological cell death exist—one that can
be inhibited by Bcl-2 and one or more that cannot (189, 198). Transgenic mice
have been generated that overexpress Bcl-2 in myeloid cells (199), neuronal cells
(200, 201), hepatocytes (175, 176), spermatogonia (202), or a relatively wide range
of cell types (203). Three transgenic animal studies of Bcl-xL were performed in
B- and T-lymphoid cells (140, 204, 205) and one in pancreatic β cells (206). As
expected, cells expressing these anti-apoptotic transgenes exhibit extended survival
in tissue culture and in the whole animal.
Gene knockout technology has been used to determine the essential functions
of Bcl-2 family proteins. Mice deficient in Bcl-2 show increased cell death during
230 STRASSER ¥ O’CONNOR ¥ DIXIT

nephrogenesis and premature demise of their mature lymphocytes (190, 207–209).

These results were expected, given that Bcl-2 is normally expressed at high levels
in these tissues (210–213). Unexpectedly, there was no detectable increase in apop-
tosis in other tissues in which Bcl-2 is known to be highly expressed, such as the
nervous system, intestine, and skin (210, 214). These results may imply functional
redundancy among the anti-apoptotic Bcl-2 family members, because Bcl-xL, for
instance, is also known to be highly expressed in some of the tissues that express
Bcl-2 (127, 128). Mice deficient in bcl-x die at embryonic day 13 and show abnor-
mally increased apoptosis in the central nervous system and in erythroid cells from
fetal liver (191). This last observation now has a molecular explanation, because
it has been shown that erythropoietin can directly induce expression of Bcl-xL via
a signaling pathway involving Stat5 (215, 216). Mice lacking Bcl-w have defec-
tive spermatogenesis (217, 218), and mice lacking A1 have accelerated neutrophil
apoptosis (219). We believe that differences in the phenotypes of mice lacking anti-
apoptotic Bcl-2 family members can be explained by distinct expression patterns
and are not the result of differences in biochemical function.
Bax, Bim, and Bid are the only pro-apoptotic Bcl-2 family members for which
studies with gene knockout analysis have been published. Cell production and cell
death appear to be normal in most somatic tissues of bax−/− mice, with the excep-
tion of mild T-cell hyperplasia in the thymus and in peripheral lymphoid organs
(220). Surprisingly, bax−/− male mice exhibit marked hypoplasia in testicular cells
and are sterile (220). This phenotypic abnormality is similar to that of mice over-
expressing Bcl-2 in spermatogonia (202) and probably reflects the dependence of
spermatid production on apoptosis of neighboring cells, rather than any funda-
mental difference in the apoptosis process in germ cells. The finding that most
physiologically induced cell deaths occur normally in bax−/− mice is consistent
with the notion that Bcl-2/Bax heterodimers are dispensable for both apoptosis and
cell survival. Cell production and cell death appear to be normal in untreated Bid-
deficient mice, but, as mentioned above, their hepatocytes have reduced sensitivity
to anti-CD95 antibodies (179). Bim is required for early mouse development and
hemopoietic cell homeostasis. It is essential for certain apoptosis signaling path-
ways, notably that induced by cytokine withdrawal, and it acts as a barrier against
autoimmune disease (220a). It is anticipated that phenotypic analyses of mice lack-
ing other pro-apoptotic Bcl-2 family members (and crosses between such animals)
will reveal individual and collective roles of these proteins in cell death control.

APOPTOSIS AND DISEASE

That abnormalities in apoptosis can play a role in disease processes is beyond

dispute. The bcl-2 gene was originally isolated as a protooncogene (221–223),
and transgenic mice overexpressing Bcl-2 in lymphocytes are clearly more prone
to tumorigenesis than control littermates (7, 8, 224, 225). On its own, bcl-2 is a
relatively weak transforming oncogene, but it synergizes potently in tumorigen-
esis with growth-promoting oncogenes, such as c-myc and pim-1 (7, 226, 227).
 APOPTOSIS SIGNALING 231

This has led to the conclusion that Bcl-2 overexpression functions in neoplastic
transformation by extending the life span of cells, thereby facilitating acquisition
of further oncogenic mutations.
Recent experiments have provided evidence that the pro-apoptotic members of
the Bcl-2 family can act as tumor suppressors. Somatic frameshift mutations in the
bax gene have been found in some cases of colon cancer with the microsatellite
mutator phenotype (228). Even more convincing is that transformation of choroid
plexus epithelial cells by transgenic expression of a truncated version of the simian
virus 40 large-T antigen (which inactivates Rb but not p53) is accelerated in bax−/−
mice (229).
Chemotherapeutic anti-cancer drugs and γ -irradiation induce apoptosis in tu-
mor cells (3, 19). Oncogenes and tumor suppressor genes that regulate cell death
influence the sensitivity of tumor cells to anti-cancer therapy. Overexpression of
Bcl-2 and its pro-survival homologs or inactivation of Bax not only provides short-
term protection against apoptosis, but can significantly increase long-term survival
with retention of clonogenicity in certain tumor cells that have been treated with
anticancer drugs or γ -radiation (230, 231). Thus, the response of cancer cells to
therapy is determined by at least two processes, the propensity to undergo mitotic
death and the sensitivity to apoptotic stimuli. Many anti-tumor therapies rely on
inducing apoptosis in their target cells. The role of caspases in the response or
resistance to such drugs has therefore been under intense scrutiny. Because the
various caspases can process each other, most of them eventually become acti-
vated in cells undergoing apoptosis, and this appears to be the case in drug-treated
tumor cells (34, 232–235). Therefore, the critical question is not which caspases
are activated in response to a particular apoptotic stimulus, but which initiator cas-
pase must be activated for apoptosis to occur. Experiments with synthetic caspase
inhibitors have yielded contradictory answers to this question. One study indicated
that caspase-1 or a closely related caspase was critical for drug-induced apopto-
sis in certain human glioma cell lines (236). In contrast, other studies concluded
that caspase-1 and caspase-3 (237), or all caspases sensitive to zVADfmk, are
dispensable for drug-induced cell death (238).
Experiments with tumor cell lines expressing virus-encoded caspase inhibitors
and studies of nontransformed cells from caspase-deficient mice have provided
clarification. CD95- and TNF-RI–induced apoptosis can be blocked specifically
by the cowpox virus serpin CrmA (137, 172, 174, 239–241), which potently in-
hibits caspases-1 and -8, but has much less effect on other mammalian caspases
(242). This property of CrmA was used to analyze the role of CD95 in drug- and
radiation-induced apoptosis. CrmA did not confer drug resistance on lymphoid
cells (94, 137, 172, 243, 244), whereas p35, the potent broad-spectrum caspase in-
hibitor from baculovirus, could protect against death receptor-induced apoptosis
as well as γ -radiation (41, 243). These results indicated that caspases other than
caspases-1 or -8 are the essential inducers of drug-induced apoptosis. Consistent
with this notion, cells from caspase-8 deficient mice are normally sensitive to
chemotherapeutic drugs and γ -irradiation (86), but those lacking caspase-9 are
highly resistant (39, 40).
232 STRASSER ¥ O’CONNOR ¥ DIXIT

Inappropriate survival of cells does not have consequences only for tumorigene-
sis. The immune response is characterized by rapid cell proliferation in response to
pathogens and equally rapid apoptotic death of responder cells after the pathogen
has been eliminated (189, 198, 245). Abnormally prolonged survival of activated
lymphocytes, which produce effector molecules that may be damaging to the host,
can have dire consequences. Overexpression of Bcl-2 in B lymphocytes of trans-
genic mice or Bim deficiency results in prolonged humoral immune responses
and pathological accumulation of plasma cells, which can eventually lead to fatal
systemic lupus erythematosus-like autoimmune disease (9, 246, 247, 220a). The
discovery that mutations in CD95 or its ligand cause lymphadenopathy and au-
toimmunity in mice (10, 248–250) and humans (251, 252) provided additional
support for the notion that the normally short life span of effector lymphocytes
constitutes a vital barrier against autoimmune attack.
Apoptosis is used as a defense mechanism against viruses and other intracellular
pathogens. In turn, many of these pathogens have developed mechanisms to inhibit
the death of host cells to promote their replication and/or persistence (253, 254).
Cells can activate the apoptotic machinery as a result of sensing metabolic distur-
bances caused by viral infection. For example, expression of adenovirus protein
E1A promotes viral replication but also triggers host cell apoptosis by activating
the tumor suppressor protein p53 (255). This pathway to apoptosis can be blocked
by two adenovirus proteins, E1B55kD, which directly interferes with p53 func-
tion, and E1B19kD, a Bcl-2 homolog, which inhibits apoptosis signaling further
downstream (256). Several other types of viruses also have Bcl-2 homologs that
presumably serve to prevent host cell apoptosis caused by stress or growth factor
deprivation (253, 254).
In addition to the Bcl-2 homologs, viruses have acquired other inhibitors of
apoptosis. The cowpox virus-encoded serpin CrmA is a potent inhibitor of some
(e.g. caspases-1 and -8) but not all caspases (41, 257, 258). By blocking caspases-1
and -8, CrmA provides several benefits to the virus. It prevents production of
interleukin-1β and interferon-γ , thereby reducing inflammation (257, 259), and it
can also inhibit apoptosis induced by CD95, p55 TNF-RI, and potentially other
death receptors (137, 239, 240). Another viral caspase inhibitor is baculovirus pro-
tein p35, which potently antagonizes all known caspases [from mammals, nema-
todes, and insects (41)] and appears to be able to block all pathways to apoptosis
in these organisms (42, 43, 45).
Cytotoxic T lymphocytes are a major defense against virus infection. On anti-
gen receptor triggering, cytotoxic T lymphocytes kill target cells by releasing
perforin plus granzymes or through the action of FasL and TNF (260–263). To
evade detection, some viruses have developed mechanisms that inhibit presentation
of virally encoded antigens by major histocompatibility complex molecules in in-
fected cells (264). Several viruses have acquired molecules that specifically inhibit
CD95- and/or p55 TNF-RI–induced apoptosis (254). Some pox viruses encode sol-
uble TNF receptor homologs (e.g. PV-A53R, CrmB, and PV-T2) that neutralize
all actions of TNF and lymphotoxin. By inhibiting apoptosis and inflammation,
they subvert the host immune response (265, 266). A complex of two proteins from
 APOPTOSIS SIGNALING 233

adenoviruses, E3-10.4kD and E3-14.5kD, mediates loss of p55 TNF-RI and CD95
from the cell surface and thereby promotes resistance to apoptotic signaling from
these two death receptors (267, 268).
Viral homologs of cellular FLIP, vFLIP molecules, have been described in some
γ -herpes viruses and pox viruses (269–272). They contain 2 DEDs but lack the
regions that give rise to the p20 and p10 polypeptides, and therefore resemble
the short form of FLIP, FLIPS. The vFLIPs bind with one of their two DED
domains to the single DED in the adaptor protein FADD, inhibit recruitment and
activation of pro-caspase-8, and thereby block apoptosis triggered by several of
the death receptors (269–271). The adenovirus protein E3-14.7kD does not have
a recognizable DED, but it can bind to caspase-8 and thereby block p55 TNF-RI-
and CD95-transduced apoptosis (273, 274).
It is interesting that many viruses carry both a Bcl-2 homolog and a specific
inhibitor of death receptor-induced apoptosis. For example, adenovirus type 5
expresses the Bcl-2 homolog E1B19kD and the E3-14.7kD protein, a specific in-
hibitor of caspase-8, and human γ -herpes virus 8 (HHV-8) expresses the Bcl-2
homolog ORF16 and the vFLIP ORF71. It only makes sense for a virus to carry
both types of inhibitors if they block distinct pathways to apoptosis. If the Bcl-2 ho-
mologs could block death receptor-induced apoptosis, there would be no selective
pressure for a virus to also express a vFLIP or E3-14.7kD protein. These con-
siderations are consistent with the idea that mammals have two distinct apoptosis
signaling pathways (Figure 5).

A MODEL FOR THE BIOCHEMICAL ACTION

OF THE BCL-2 FAMILY
We believe that there are two major gaps in our understanding of how apoptosis is
controlled at the molecular level. First, it is unclear how anti-apoptotic members
of the Bcl-2 family prevent caspase activation by adaptor molecules such as Apaf-
1 and Ced-4. Second, it is unclear how the anti-apoptotic members of the Bcl-2
family are themselves regulated by the two different types of pro-apoptotic Bcl-2
family members.
The findings described in this review are consistent with the following model
for the control of caspase activation by the Bcl-2 family. Cell death signals lead
to the activation of both Ced-4–like proteins and pro-apoptotic members of the
Bcl-2 family (134). Survival signals, such as those from cytokine receptors, can
increase the levels of anti-apoptotic Bcl-2 family members (141) and can reduce
the activity of the pro-apoptotic Bcl-2 family members, probably by sequestering
them to sites where they cannot interact with anti-apoptotic Bcl-2 family mem-
bers (146, 147, 151). Survival signals may also have effects on the Ced-4–related
proteins. Ced-4 and its homologs act as adaptors for caspase zymogens and are
thought to trigger their conversion into the active tetrameric enzyme complex.
Bcl-2 and its homologs promote survival by binding to and blocking the action of
Ced-4–related proteins. It is likely that this interaction takes place in a multiprotein
234 STRASSER ¥ O’CONNOR ¥ DIXIT

complex that contains additional regulators. The AAA ATPase MAC-1, which has
been found to associate with Ced-4 and Ced-9 and whose overexpression can pre-
vent programmed cell death in C. elegans, may be such an additional regulator
(275). The pro-apoptotic Bcl-2 family members prevent binding of anti-apoptotic
Bcl-2 family members to Ced-4 or its homologs (157). Thus, apoptosis is induced
by inhibiting the activity of proteins that inhibit activators of the caspases.
However, at least two alternative models to explain the function of the Bcl-2
protein family have been proposed. The three-dimensional structure of Bcl-xL
showed some structural similarities to pore-forming bacterial toxins (276), and
studies in synthetic lipid membranes have shown that Bcl-2, Bcl-xL, and Bax
can form ion channels, albeit mostly at nonphysiological pH (277–279). Many
Bcl-2 family members have a conserved C-terminal transmembrane region that is
responsible for their localization to the cytosolic aspect of the nuclear envelope,
outer mitochondrial membrane, and endoplasmic reticulum (280, 281). This led to
the idea that anti-apoptotic and pro-apoptotic members of the Bcl-2 family function
as transmembrane channels that promote or hinder the efflux of molecules that
cause caspase activation.
Biochemical experiments with cell lysates have shown that four molecules
[apoptotic protease-activating factors (Apafs)] are required for the processing of
caspase-3 zymogens in vitro: dATP, Apaf-1 (the mammalian homolog of Ced-4),
Apaf-3 (caspase-9), and cytochrome c (183, 184, 282). The pro-survival proteins
Bcl-2 and Bcl-xL have been shown to inhibit the release of cytochrome c from
mitochondria into the cytosol of dying cells, whereas pro-apoptotic Bcl-2 family
members were shown to promote this process (21, 22, 183). These observations
have led to a model of Bcl-2 family function in which apoptotic stimuli cause
disturbances in mitochondria that lead to release of cytochrome c and consequently
to caspase activation, and Bcl-2 and its homologs function by maintaining the
integrity of the mitochondria. There is some supporting evidence for this model,
including the observation that Bcl-2 family members can regulate cytochrome c
release by directly binding to components of the voltage-dependent anion channel
in mitochondria (283). This model does not, however, account for the finding
that adenovirus protein E1B19kD, which is localized exclusively to the nuclear
envelope and the endoplasmic reticulum (284), inhibits apoptosis as well as Bcl-2
or Bcl-xL (138).
We favor the first of these three models for explaining the molecular control of
apoptosis and envisage that the other two processes may be used to amplify the
caspase cascade after the death process has been initiated (186).
We present here a novel speculative model for biochemical regulation of apop-
tosis by the Bcl-2 family. We have been led to this speculation by a number of
puzzling facts, some of which have arisen from studies of Ced-9, the C. elegans
Bcl-2 homolog. Although C. elegans lacking Ced-9 have increased cell death, Ced-
9 was found to actually promote apoptosis in the context of mutant Ced-3, whose
activity was compromised (104). Furthermore, mutation of a single amino acid
has the effect of increasing the anti-apoptotic potency of Ced-9 (ced-9gf) (285),
whereas mutation of the corresponding residue in Bcl-2, Bcl-xL, or Bcl-w destroys
 APOPTOSIS SIGNALING 235

anti-apoptotic function (97, 140, 286). These observations suggest that Ced-9 can
have anti-apoptotic properties under some circumstances and pro-apoptotic prop-
erties under others. We have also been puzzled by the observation that there are
mutants of Bcl-xL that are incapable of binding to pro-apoptotic Bcl-2 family mem-
bers, but retain their pro-survival function (115, 287). This suggests that binding
to pro-apoptotic Bcl-2 family members is not necessary for anti-apoptotic proteins
to function, but rather that the pro-apoptotic proteins must alter the anti-apoptotic
proteins in some way to facilitate caspase activation and promote apoptosis. An-
other piece of information has arisen from the analysis of mice lacking the pro-
apoptotic BH3-only protein Bim. Lymphoid cells have <1000 Bim molecules, and
Bim-deficient lymphoid cells have strikingly similar abnormalities to those from
Eµ–bcl-2–transgenic mice, which express ∼200,000 Bcl-2 molecules (220a). This
suggests that the number of Bim molecules needed to induce apoptosis is much
less than the number of anti-apoptotic molecules needed for cell survival. This
contradicts the so-called rheostat model of Bcl-2 family regulation, where pro-
and anti-apoptotic Bcl-2 family members titrate each other in roughly equal stoi-
chiometry (119), and suggests that a large number of anti-apoptotic molecules can
be altered in some way by seeding with a few pro-apoptotic molecules.
Here, then, is a possible explanation for the above observations. Anti-apoptotic
Bcl-2 family members such as Bcl-2 are found in membranes and may form a large
macromolecular structure or lattice. The conformations of individual proteins in
this lattice are affected by their neighbors and may be induced to switch between
two states; pro-apoptotic and anti-apoptotic. When a pro-apoptotic member of the
Bcl-2 family binds to an anti-apoptotic member, it induces it to switch confor-
mation. In their switched state, these proteins then induce nearby anti-apoptotic
molecules to switch their conformation, and the entire lattice becomes conducive
to caspase activation and apoptosis induction (Figure 6). This model is analogous
to the currently accepted model of prion function, in which mutant prion molecules
can change the conformation of normal prion molecules, which then change the
conformation of their normal neighbors when they are juxtaposed in the plasma
membrane of cells (288).
This model answers the questions raised above. The anomalous behavior of
Ced-9gf in the context of mutant Ced-3, and the contradictory effect of equivalent
mutations in mammalian pro-survival proteins, can be reconciled if pro-survival
proteins are thought of as capable of being switched to a pro-apoptotic state by
binding to a pro-apoptotic Bcl-2 family member. In C. elegans, if a mutant form of
Ced-9 is incapable of being switched to a pro-apoptotic state, activation of Egl-1
will have no effect; Ced-9gf, which does not bind to Egl-1, appears to be such a
mutant. In mammalian cells, however, there are several pro-survival Bcl-2 family
members, so activation of pro-apoptotic Bcl-2 family members may still switch
endogenous pro-survival molecules and trigger apoptosis, despite overexpression
of mutant pro-survival molecules that cannot bind to pro-apoptotic proteins. The
existence of Bcl-xL mutants that protect cells from apoptosis but cannot bind to Bax
or Bim is also explained, because these Bcl-xL mutants may be refractory to being
switched to a pro-apoptotic state and would thus function by stabilizing the lattice
236 STRASSER ¥ O’CONNOR ¥ DIXIT

Figure 6 A speculative model of Bcl-2 family regulation in which a lattice of anti-apoptotic Bcl-
2 family members (blue symbols) can be switched between pro-apoptotic (orange rectangles) and
anti-apoptotic conformational states by the binding of a few pro-apoptotic Bcl-2 family members.

in an anti-apoptotic state. This model also explains the different potencies of the
two classes of pro-apoptotic Bcl-2 family members. Pro-apoptotic Bcl-2 family
members with several BH domains, such as Bax, are not as potent at inducing
apoptosis as BH3-only proteins, such as Bim. BH3-only proteins may disrupt the
conformation of anti-apoptotic Bcl-2 family members very efficiently, whereas the
Bax-like pro-apoptotic Bcl-2 family members may merely mimic anti-apoptotic
Bcl-2 family members in their switched state. It can also be seen that very few pro-
apoptotic molecules would be needed to seed an intracellular membrane and make
it conducive to caspase activation, which explains why the loss of a small number
of endogenous Bim molecules is equivalent to overexpression of a large number
of Bcl-2 molecules. We stress that this model is speculative and meant merely as
an interesting hypothesis to stimulate further discussion and investigation.

CONCLUSIONS AND PERSPECTIVE

It has been established that apoptotic cell death plays an essential role in normal
development and functioning of multicellular organisms and that abnormalities in
this process can cause disease. Some understanding of the molecular control of
 APOPTOSIS SIGNALING 237

apoptosis has been gained, but the jigsaw puzzle is incomplete. The pace in cell
death research is frantic and, sadly, this has resulted in considerable confusion
caused by a lot of contradictory publications. Unfortunately, many conclusions
on signal transduction, protein-protein interaction, and protein function are based
solely on data from overexpression systems in transformed cells. Because dysreg-
ulated cell death control can cause cancer (7, 289), we must be wary of cultured
tumor cell lines having unidentified mutations in cell death regulators. More em-
phasis should therefore be given to those studies on gene function and signal trans-
duction that are performed in nontransformed cells. Also, to be able to generate
models for cell signaling that are relevant to normal cell physiology, protein-protein
interactions must be confirmed in cells in which all components are expressed at
physiological levels. We also believe that a more complete understanding of cell
death control is needed before we contemplate designing therapies that modulate
the apoptotic effector machinery.

ACKNOWLEDGMENTS
Current work by the authors is supported by Genentech Inc. (VMD), by the Na-
tional Health and Medical Research Council (Canberra, Australia), the Dr. Josef
Steiner Cancer Foundation (Bern, Switzerland), a Clinical Investigator Award from
the Cancer Research Institute (New York, NY), the Leukemia Society of America
(AS), and the Anti-Cancer Council of Victoria (AS and LO’C.). We thank all of our
past and present colleagues, particularly Drs. J Adams, S Cory, A Harris, D Vaux,
J Miller, J Allison, K Newton, H Puthalakath, L O’Reilly, A Villunger, P Bouillet,
and D Huang for stimulating discussions and their input into our work, and we are
grateful to J Birtles for editorial assistance. We apologize to those scientists in the
field whose work was mentioned in the references only indirectly through reviews.

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