Biochimica et Biophysica Acta 1551 (2001) F1^F37

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Review
Apoptosis regulators and their role in tumorigenesis
a;
Martin Zo«rnig *, Anne-Odile Hueber b , Wiebke Baum a , Gerard Evan c

a
Georg-Speyer-Haus, Paul-Ehrlich-StraMe 42^44, 60596 Frankfurt, Germany
b
Institute of Signaling, Developmental Biology and Cancer Research CNRS UMR 6543, Centre A. Lacassagne, 33 Avenue Valombrose,
06189 Nice, France
c
UCSF Cancer Center, 2340 Sutter Street, San Francisco, CA 94143-0128, USA

Received 9 May 2001; received in revised form 12 July 2001; accepted 25 July 2001

Abstract

It has become clear that, together with deregulated growth, inhibition of programmed cell death (PCD) plays a pivotal role
in tumorigenesis. In this review, we present an overview of the genes and mechanisms involved in PCD. We then summarize
the evidence that impaired PCD is a prerequisite for tumorigenesis, as indicated by the fact that more and more neoplastic
mutations appear to act by interfering with PCD. This has made the idea of restoration of corrupted `death programs' an
intriguing new area for potential cancer therapy. ß 2001 Elsevier Science B.V. All rights reserved.

Keywords: Apoptosis; Tumorigenesis; Programmed cell death

1. Introduction cytes to delete autoreactive or non-reactive cells,

For many biologists it came as a surprise to realize
that the death of a cell is not necessarily a bad thing.
Indeed, in metazoans cell death is required for devel-
opment, maintenance and survival of the organism.
Physiological cell death has been observed in di¡er-
ing tissues and in various organisms for more than
100 years [1]. Cell death occurs throughout the life
span of multicellular organisms and arguably repre-
sents the only irreversible cell fate decision. Promi-
nent examples of physiological apoptosis include the
hormonally regulated involution of the tadpole tail
during development, negative selection of lympho-
* Corresponding author. Tel.: +49-69-63395115;
Fax: +49-69-63395297.
E-mail address: zoernig@em.uni-frankfurt.de (M. Zo«rnig).
widespread cell death of neuronal cells during the
self-assembly of the central nervous system, and the
formation of digits by involution of interdigital cells
in the primitive limb paddle (a more extensive survey
of literature describing apoptosis occuring in vivo
can be found in [2]).
Apoptosis is, by far, the most predominant form
of physiological cell death. In contrast, unambiguous
examples of physiological cell necrosis are few. Be-
cause it is a regulated process, controlled by a diver-
sity of extracellular and intracellular signals, apopto-
sis is used for the coordinated death of excess,
hazardous or damaged somatic cells. Moreover, the
apoptotic process includes mechanisms that organize
both packaging and disposal of cell corpses, thereby
preventing in£ammation of the surrounding tissue.
This is an essential requirement in metazoans which,
for obvious reasons, need to be able to distinguish
cells that die as part of the normal process of main-

0304-419X / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 1 9 X ( 0 1 ) 0 0 0 3 1 - 2

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F2 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37

taining tissue homeostasis from cells that die as a

result of trauma.
Severe disturbance of homeostasis of any particu-
lar cell population or lineage can cause major path-
ologies in multicellular organisms. Not surprisingly,
therefore, substantial evidence indicates that altera-
tions in control of cell death/survival contribute to
the pathogenesis of many human diseases [3]. Dis-
eases linked with suppression of apoptosis include
cancer, autoimmune disorders (e.g. systemic lupus
erythematosus) and viral infections (e.g. herpesvi-
ruses, poxviruses, adenoviruses); diseases in which
increased apoptosis is an element include AIDS [4],
neurodegenerative disorders [5], myelodysplastic syn-
dromes, ischemic injury (e.g. stroke, myocardial in-
farction), toxin-induced liver disease (e.g. alcohol)
and some autoimmune disorders [6]. In many cases,
it is unclear whether perturbations in cell death
mechanisms are causal or merely a consequence of
the disease process. Nonetheless, e¡orts aimed at
treating these diseases by manipulating cell suicide
would seem to have great potential, although they
are thus far at a relatively early stage.

Fig. 1. Cells undergoing apoptosis. (A, B) Rat-1 ¢broblasts ex-

2. Cell death: de¢nition and signi¢cance
Historically, apoptosis was de¢ned on a morpho-
logical basis by contrast with another type of cell
death, necrosis ([7,8]. The necrotic cell swells and
its chromatin takes on the appearance of a £occulent
mass that eventually disappears to leave a nuclear
ghost. Cell DNA is non-speci¢cally degraded and
characteristically appears as a smear when size frac-
tionated on an agarose gel. One of the most prom-
inent features of apoptosis involves the nucleus.
Chromatin condenses and forms aggregates near
the nuclear membrane which, in turn, becomes con-
voluted, whilst the nucleolus becomes enlarged and
appears abnormally granular. Chromatin is also sub-
ject to the actions of di¡erent activated endonu-
cleases that cleave the DNA initially into 300^50
kb fragments and subsequently into 180 bp frag-
ments [9^11]. Also during apoptosis, the cell visibly
shrinks, adherent cells round up, and distinct protu-
berances or membrane blebs become discernible (see
Fig. 1). Blebbing cells still exclude vital dyes indicat-
ing membrane integrity. Organelles within the
pressing c-Myc in the presence (A) or absence (B) of serum.
(A) Normal growing cells. (B) Rat-1 cell in a late stage of apo-
ptotic cell death. Cell shrinkage, nuclear condensation and ¢nal
fragmentation of the whole cell are obvious.
shrunken apoptotic cytoplasm retain a largely nor-
mal appearance save for some dilation of the endo-
plasmic reticulum (ER) and swelling of the mito-
chondria. The transition from normal to shrunken
and blebbing is rapid, typically taking only some
10^30 min, and it is at this point that apoptotic cells
are probably phagocytosed in vivo, either by their
nearest neighbors or by professional macrophages
[12]. Recognition and phagocytosis of apoptotic cells
is mediated by a variety of independent receptor^li-
gand interactions which will not be discussed in de-
tail further and have been reviewed elsewhere [13].
However, one feature of apoptotic cells involved in
their phagocytosis is commonly used as a marker for
apoptosis: activation of a £ippase in apoptotic cells
leads them to express externalized phosphatidylser-
ine, usually present only in the internal lea£et of
the plasma membrane [14]. The rapidity of the apo-

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 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
ptotic program and of the clearance of apoptotic
cells within the soma largely explains why apoptosis
was, until recently, largely overlooked as a major
homeostatic process.
Another morphologically distinct form of pro-
grammed cell death (PCD) is the process of auto-
phagy, or bulk degradation of cellular proteins
through an autophagosomic^lysosomal pathway.
Autophagy is important in normal growth control,
regulated by steroids during development and may
be defective in tumor cells [15,16].
3. Evolution of PCD: the nematode Caenorhabditis
elegans as an invertebrate model
Physiological PCD has been described in all multi-
cellular organisms so far studied, including plants,
slime molds, nematodes, insects and vertebrates
[17]. While the physiological role of apoptosis in
the shaping and rebuilding of complex tissues of mul-
ticellular organisms is plain to see, the biological ra-
tionale for PCD is less clear for single cell organisms.
Furthermore, unicellular PCD seems not to involve
the action of caspases ^ the cysteine proteases that
are the hallmarks of metazoan apoptosis. Nonethe-
less, there is growing evidence that some form of
PCD does exist in unicellular organisms like Trypa-
nosoma cruzi, Trypanosoma brucei rhodesiense, Dic-
tyostelium discoideum or Tetrahymena thermophila
[18] and perhaps even in bacteria [19]. Possibly,
PCD arose in unicellular organisms as a way of en-
suring survival of at least some members of a clonal
colony during periods of privation. An alternative
idea is that unicellular PCD evolved as a defense
against the spread of virus infection. Indeed, many
metazoan viruses actively suppress apoptosis in order
to ensure their propagation, indicating that host cell
suicide is an e¡ective way of forestalling virus spread.
The various known anti-apoptotic viral genes possess
a variety of di¡ering structures and modes of action.
C. elegans is well suited for the study of cell death
at the cellular, genetic, and molecular levels because
it is both transparent and developmentally invariant.
This has permitted the complete lineage description
of every one of the 1090 cells born during develop-
ment of the hermaphrodite form. During C. elegans
development, 131 cells die to leave a ¢nal total of 959
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F3
in the adult. Such detailed knowledge of develop-
mental cell deaths enabled easy identi¢cation of mu-
tants with aberrant patterns of cell death [20]. Their
analysis has identi¢ed genes controlling four aspects
of the cell death process: (1) a determination step, (2)
the execution of cell death, (3) engulfment of the
dying cell, and (4) degradation of the engulfed cell
DNA.
Three genes are involved in the critical cell death
execution step. Two of these, ced (cell death defec-
tive)-3 and ced-4 are required for each cell death [21]:
if inactivated by mutation, none of the 131 normal
cell deaths occur. The other gene, ced-9, acts to an-
tagonize the killing activity of ced-3 and -4: gain of
function ced-9 mutants show absence of cell death
whereas mutations that inactivate ced-9 lead to wide-
spread and lethal embryonic death [22]. In the last
few years, many of the molecular functions of the
proteins encoded by the ced-3, -4 and -9 genes have
been deduced [23]. The killer gene ced-3 encodes the
Ced-3 protein, a member of a class of cysteine pro-
tease that cleave after aspartate residues ^ hence their
name `caspase' (cysteine aspartyl protease [24]). Cas-
pases are synthesized as inactive zymogens that are
activated by cleavage at sites that themselves con-
form to canonical caspase cleavage sites. Activation
abscises an N-terminal prodomain and cleaves the
remaining polypeptide into small and large subunits
that then assemble as an K2 L2 active tetramer. The
Ced-4 protein physically interacts with both Ced-3
[25,26] and Ced-9 [26^29] proteins and appears to
act as an adapter protein that facilitates Ced-3 au-
toactivation [30]. Ced-4 has a putative ATPase (nu-
cleotide binding) domain that is required for its abil-
ity to activate Ced-3. The interaction of Ced-4 with
the death-inhibiting protein Ced-9 inhibits its ability
to activate Ced-3.
As discussed below, the Ced-3, -4 and -9 basal
apoptotic machinery is highly conserved amongst
the metazoa. The Ced-3 caspase is homologous to
similar enzymes identi¢ed in insects and in verte-
brates ^ the prototypical mammalian homologue
being the eponymous interleukin-1L converting en-
zyme (ICE) [31], although some 14 other mammalian
caspases are now known [32,33].
The mammalian homologues of Ced-9 are the Bcl-
2 proteins [34,35] ^ key regulators of cell survival
¢rst identi¢ed by the oncogenic activity of Bcl-2 in
F4 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
human follicular B cell lymphoma. Recent evidence
suggests that Ced-9 prevents apoptosis in C. elegans
by two distinct mechanisms [36]: it may directly in-
hibit the Ced-3 protease by an interaction involving
Ced-3 cleavage sites within Ced-9, or Ced-9 may also
directly or indirectly inhibit Ced-3 by means of a
protective mechanism similar to that used by mam-
malian Bcl-2: cleavage of Ced-9 by Ced-3 generates a
carboxy-terminal product that resembles Bcl-2 in se-
quence and in function.
A mammalian homologue of Ced-4 has been iden-
ti¢ed as Apaf-1 (apoptotic protease activating fac-
tor), a large protein implicated in regulating caspase
activity through mediating cytochrome c-dependent
activation of caspase-9 [37]. Such tremendous evolu-
tionary conservation of cell death machinery
amongst metazoans has the bene¢t that analysis of
cell death in `simple' genetic model systems like
C. elegans is very informative in developing our
understanding of the control of apoptosis in mam-
malian cells. For example, the C. elegans egl-1 en-
codes a small protein containing a nine amino acid
stretch similar to the BH3 domain, a domain found
in various pro-apoptotic Bcl-2-like mammalian cell
death regulators. Experimental analysis showed that
egl-1 probably acts upstream of ced-3 and ced-4, and
direct interaction between the Egl-1 and Ced-9 pro-
teins was also demonstrated [38]. This led to the
suggestion that Egl-1 may act by interfering with
Ced-9 so that it can no longer inhibit Ced-4, which
is then free to activate Ced-3, leading to cell death.
Further genes required for removal of apoptotic
cells have been identi¢ed in C. elegans [20] resulting
in mammalian homologues being identi¢ed and char-
acterized. The recently cloned human homologue of
the C. elegans engulfment protein Ced-6, for exam-
ple, speci¢cally promotes phagocytosis of apoptotic
cells [39,40].
4. Molecular pathways of cell death
4.1. Central e¡ectors: caspases
It is widely accepted that caspases, the family of
related Ced-3-like cysteine proteases, are common
e¡ectors of classical metazoan apoptosis. The ¢rst
mammalian homologue of Ced-3 identi¢ed in 1993
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
was ICE [31], now called caspase-1. Caspase-1 and -
11 (and possibly -4 and -5) are principally involved in
proteolytic maturation of cytokines such as interleu-
kin-1L [41^43] and may have little to do with apo-
ptosis. However, subsequent studies have identi¢ed a
further 10 ICE family members of which caspases-2,
-3, -6, -7, -8, -9, -10, -12, -13 and -14 are all impli-
cated in regulation and/or execution of apoptosis:
Nedd-2/ICH-1 (caspase-2), Yama/CPP32/apopain
(caspase-3), Tx /ICH-2/ICErelII (caspase-4), ICErelIII
(caspase-5), Mch-2 (caspase-6), ICE-Lap-3/Mch-3/
CMH-1 (caspase-7), FLICE/MACH (caspase-8),
ICE-LAP-6 (caspase-9), Mch-4/FLICE-2 (caspase-
10), ICH-3 (caspase-11), caspase-12, ERICE (cas-
pase-13) and MICE (caspase-14) [32,44].
These apoptotic caspases undergo activating cleav-
age during apoptosis (either through autoactivation,
as part of a caspase cascade or by other non-caspase
proteinases such as granzyme B) and between them
they cut a range of substrate proteins whose cleavage
either mediates or attends the apoptotic process. Fur-
thermore, caspase inhibitors, whether virus proteins
such as cowpoxvirus CrmA or baculovirus p35, or
aldehydes or £uoromethyl ketone-derivatized syn-
thetic peptide inhibitors based on preferred substrate
sequences (e.g. ZVAD-fmk), suppress many aspects
of mammalian apoptosis.
Caspases share many common structural and cat-
alytic features. All contain an active site pentapeptide
sequence with the general structure QACXG (where
X is R, Q or G): the cysteine (together with a distant
histidine) is directly involved in catalysis. In the
main, caspases recognize a tetrameric primary se-
quence in their substrates with a distinctive require-
ment for an aspartic acid residue in the substrate
P1 position. As tetrapeptides corresponding to the
substrate P4 ^P1 residues are su¤cient for speci¢c rec-
ognition by caspases [45], such substrates have pro-
vided the basis for design of a range of inhibitors
[46]. Phylogenetic analysis of the caspases shows
they fall into three subfamilies, each with signi¢-
cantly di¡ering substrate speci¢city which generally
correlates with caspase function: the ICE subfamily
of cytokine processors (caspases-1, -4, -5 and -11;
because of sequence homology to caspase-1 the cas-
pases-12, -13 and -14 are also grouped with the cy-
tokine processors), the Ced-3/CPP32 subfamily of
apoptotic executioners (caspases-3, -6, and -7) and
 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
the ICH-1/Nedd-2 subfamily of apoptotic initiators
(caspase-2, -8, -9 and -10).
Caspases are synthesized as precursor proenzymes
which are proteolytically processed to their active
forms. Active caspases are composed of a hetero-
dimer comprising a large subunit (P20 for caspase-
1, P17 for caspase-3) that contains the catalytic cys-
teine residue, and a smaller subunit (P10 for caspase-
1, P12 for caspase-3) that contains determinants
which govern substrate speci¢city. The X-ray crystal
structure of activated caspase-1 indicates that two
independent functional P20/P10 heterodimers are in-
timately associated to form a (P20/P10)2 tetramer in
which the two active sites reside at opposite ends of
the complex [47,48]. Procaspases are activated by
cleavage at critical aspartate residues that themselves
conform to the substrate consensus for caspases.
This indicates that caspases exist within hierarchies
of families that undergo auto- and trans-cleavage.
For example, evidence supports the idea that cas-
pase-8 autoactivates itself upon recruitment to a
death receptor signaling complex (the zymogene full
length caspase-8 molecule obviously possesses some
residual proteolytic activity su¤cient for this self-
cleavage) and the active enzyme then in turn cleaves
and activates the e¡ector caspases-3 and -7 that ex-
ecute the apoptotic program. Interestingly, caspase-9
does not necessarily require proteolytic processing,
but instead requires binding to APAF-1 with which
it forms an active holoenzyme [49,50].
All procaspases have an N-terminal prodomain
that is also removed during activation. For some
caspases (caspase-3, -6, -7 and -14) the prodomain
is short (10^40 residues) whilst for the other caspases
it is extensive and contains recognizable domains.
The extensive prodomains play important roles in
caspase regulation and function as signal integrators
for apoptotic or pro-in£ammatory signals [51,52].
For example, the prodomains of caspase-8 and -10
each contain two so-called death e¡ector domains
that mediate the proenzymes' interaction, via an
adapter molecule, with the cytoplasmic tail of mem-
bers of the TNF-R1/CD95 receptor family. This al-
lows receptor-induced activation of the caspases in
response to ligand binding. Caspases-1, -2, -4, -9
and Ced-3 possess a distinct prodomain structure
termed CARD (caspase recruitment domain).
CARD domains (also found in the adapter protein
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F5
APAF-1) are presumed to mediate speci¢c intermo-
lecular interactions that regulate caspase activation.
Indeed, one strongly inactivating mutation of Ced-3
is located within its prodomain [53], indicating the
importance of this region for Ced-3 activation.
CARDs and death e¡ector domains as well as death
domains, all contain six anti-parallel K-helices ar-
ranged in a similar three-dimensional fold and asso-
ciate via like^like interactions [32].
Genetic knockout studies in mice have been used
to investigate the measure of redundancy amongst
caspases [54]. Available data suggest that apoptosis
triggered by di¡ering stimuli frequently employ dif-
ferent `subsets' of activated caspases. Thus, mice de-
¢cient in caspase-1 develop normally, are fertile, ap-
pear healthy and exhibit no apparent abnormalities,
suggesting absence of any gross defects in normal
physiological processes involving apoptosis [41,55].
However, thymocytes from the caspase-1null mice ex-
hibit partial resistance to CD95-induced apoptosis,
implying a role for caspase-1 in implementing apo-
ptosis in response to that particular trigger. Caspase-
11 knockout mice also show defective interleukin-1L
production but develop normally and have minimal
apoptotic defects [43].
Mice lacking caspase-3 are smaller than their wild-
type littermates and die at 1^3 weeks of age [56].
Analysis shows that the development of the brain
in these animals is markedly a¡ected: a variety of
central nervous system hyperplasias are observed
from embryonic day 12 on, indicative of defective
apoptosis. However, thymocytes from caspase-3null
mice exhibit normal sensitivity to apoptosis induced
by a number of di¡erent stimuli and the rather tis-
sue-restricted phenotype in caspase-3-de¢cient mice
again demonstrates that other caspases can substitute
for caspase-3 in most tissue or cell types. Animals
de¢cient in caspase-8 or caspase-9 die perinatally be-
cause of profound defects in developmental cell
deaths [57^59]. Caspase-2-de¢cient mice develop nor-
mally, but cells from these animals show diminished
or enhanced apoptosis, depending on their tissue of
origin [60]. Caspase-12 is localized to the ER and
becomes activated by ER stress. Mice that are de¢-
cient in caspase-12 are resistant to ER stress-induced
apoptosis, but their cells undergo apoptosis in re-
sponse to other death stimuli [61].
Caspase substrates [62,63] can be grouped into dif-
F6 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
ferent classes according to their (putative) function.
Amongst these are pro- and anti-apoptotic proteins,
components of the apoptotic machinery, structural
proteins, homeostatic proteins and proteins impor-
tant for signaling, cellular repair and macromolecu-
lar synthesis [32]. The following examples represent
these di¡erent classes of caspase substrates. (1)
Transactivation of procaspases by already activated
caspases could generate su¤cient proteolytic activity
to overwhelm endogenous caspase inhibitors such as
inhibitor of apoptosis proteins (IAPs). (2) Caspase-3
cleaves Bcl-2 and Bcl-xL which destroys the anti-ap-
optotic function of these proteins and releases C-ter-
minal fragments that are pro-apoptotic [64,65]. (3)
Caspase-8 cleaves Bid, a pro-apoptotic Bcl-2 family
member, generating a C-terminal fragment that in-
duces release of mitochondrial cytochrome c [66,67].
(4) Caspase-3 cleaves ICAD/DFF45 allowing the
ICAD-bound nuclease CAD to translocate to the
nucleus and to cut DNA [68^70]. (5) Caspase-3
also cleaves and activates Gelsolin, a protein that
regulates actin dynamics and promotes both cyto-
plasmic and nuclear apoptosis, including DNA frag-
mentation [71]. (6) Lamins are major structural pro-
teins within the nuclear envelope, and their cleavage
by caspase-6 may be responsible for some of the
observed nuclear changes [72]. (7) Cleavage of L-cat-
enin and FAK might interrupt cell^cell contacts and
cell^matrix focal adhesions thereby promoting cellu-
lar packaging and phagocytosis [73,74]. (8) Poly(-
ADP-ribose) polymerase (PARP) cleavage may inter-
fere with its key homeostatic function as a DNA
double-strand break repair enzyme [75] which might
facilitate or allow the DNA degradation character-
istic of apoptosis [33]. However, it is worth noting
that PARPnull mice seem to develop normally [76].
Unfortunately, in the case of many caspase sub-
strates it is not always obvious what, if any, mecha-
nistic role their cleavage plays in apoptosis. Many
caspase substrates are `plausible' candidate e¡ectors
for apoptosis, but so far no `key' target has been
found whose cleavage appears to provide the ulti-
mate `killer' cut for the cell. Nonetheless, given the
central role of caspases in the apoptotic program,
these proteases o¡er obvious therapeutic targets for
the control of inappropriate apoptosis [46]. Caspase
over-reactivity promotes cellular suicide, and this
may be the basis for degenerative disorders such as
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
Huntington's disease and Alzheimer's disease: cas-
pase-3 and -12 seem to be involved in proteolytic
cleavage of Alzheimer amyloid-L precursor protein
and formation of the apoptosis-inducing amyloido-
genic AL peptide [61,77]. The polyglutamine repeats
associated with Huntington's disease induce neuronal
cell death via caspase-8 [78]. Caspase-10 loss of func-
tion mutations have been linked to defective cell
death in autoimmune lymphoproliferative syndrome
type II [79]. Caspase inactivation may also promote
oncogenesis [80]. However, there is one caveat: some
triggers of apoptosis retain the ability to kill cells
even when caspases are inhibited [81^84], although
it is not clear what mechanisms are involved in
such caspase-independent cell death. The CD95/
Fas/Apo-1 receptor is able to kill activated primary
T-cells in the absence of active caspases (see below);
this cell death involves necrotic morphological
changes and depends on the kinase Rip as e¡ector
molecule [85].
4.2. The ancestral pathway: the role of mitochondria,
cytochrome c and Apaf-1 in mammalian apoptosis
Ideas concerning the mechanism of activation and
control of apoptosis have been greatly in£uenced by
the recent discovery that cytochrome c is released
from mitochondria during cell death and is involved
in triggering the e¡ector machinery of apoptosis
[23,86^89]. Cytochrome c normally resides in the
space between the outer and inner membranes of
mitochondria where it participates in the process of
oxidative phosphorylation. Upon exposure of cells to
apoptotic stimuli, cytochrome c is released from mi-
tochondria into the cytosol, where it is one of several
factors implicated in the proteolytic activation of
caspase-3 by caspase-9 [90].
Biochemical analysis has identi¢ed two cytosolic
proteins, Apaf-1 and Apaf-3, that form the complex
with cytochrome c that activates caspase-3. Apaf-1
shares limited homology with the product of the
C. elegans ced-4 gene product [37], although it is a
larger and more complex protein, while Apaf-3 is
caspase-9 [91]. The Ced-4-like domain in Apaf-1 is
£anked on one side by a region with strong homol-
ogy to the CARD motif within the prodomains of
Ced-3 and mammalian caspases-2 and -9 and on the
other side by several WD-40 repeats believed to me-
 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
diate protein^protein interactions. The CARD do-
mains in Apaf-1 and the prodomain of caspase-9
interact and, in the presence of cytochrome c and
either ATP or ADP, this induces autocatalytic acti-
vation of the caspase which then activates the down-
stream caspase e¡ector cascade involving caspases-2,
-3, -6, -7, -8 and -10 [90].
Apaf-1 dimerization is repressed by its own C-ter-
minus containing the WD-40 repeats. It is speculated
that cytochrome c binding to the WD repeats then
induces a conformational change that allows Apaf-1
to oligomerize and by promoting clustering of this
caspase to activate caspase-9 [92].
The Apaf-1/caspase-9 interaction is clearly reminis-
cent of the mechanism of Ced-3 activation by Ced-4
in C. elegans. This similarity extends to the involve-
ment of the mammalian anti-apoptotic Bcl-2 and
Bcl-xL , proteins which are homologues of Ced-9.
Bcl-2/Bcl-xL reside in the outer mitochondrial mem-
brane, where they function to suppress apoptosis in
both or either of two ways: blocking cytochrome c
release and binding to Apaf-1 to prevent its activat-
ing caspase-9. The mammalian pro-apoptotic Bcl-2
family members, such as Bax, Bak and Bik, may
promote apoptosis by displacing Apaf-1 from Bcl-
2/xL . Nevertheless, although a direct binding of
Bcl-xL to Apaf-1 (shown by in vitro experiments)
has been reported [93,94], these data (or at least their
physiological relevance) have been questioned re-
cently [95]. The anti-apoptotic protein Aven, which
was identi¢ed in a yeast two-hybrid screen, has been
shown to bind to both Bcl-xL and Apaf-1 and this
molecule might link the two molecules and target the
Bcl-2 family member to the apoptosome [96]. The
fact that direct Bcl-xL or Bcl-2 binding to Apaf-1
could not be observed in vivo might also indicate
that other Ced-4 homologues exist which could
bind to anti-apoptotic Bcl-2 family members. The
idea of further Ced-4 family members is supported
by the murine Apaf-1 knockout which leads to an
impairment of apoptosis in some, but not all circum-
stances and cell types [97,98]. Targeted inactivation
of Apaf-1 in mice nevertheless leads to profound
developmental abnormalities in cell number regula-
tion in the brain as well as in other tissues such as
the peripheral nervous system, resulting in embryonic
lethality.
By using green £uorescent protein (GFP)-tagged
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F7
cytochrome c transfected into HeLa cells it was re-
cently demonstrated that the release of cytochrome
c-GFP always precedes exposure of phosphatidylser-
ine at the cell surface and the loss of plasma mem-
brane integrity [99]. The time interval between death
stimulus and cytochrome c release can vary in indi-
vidual cells (and depending on the apoptotic insult)
but once initiated, cytochrome c is released from all
mitochondria in individual cells within 5 min. This
study also showed that the drop in the mitochondrial
membrane potential typically seen in apoptotic cells
occurs later than cytochrome c release from mito-
chondria and that this process is dependent on cas-
pase activation, whereas cytochrome c release is not.
These results suggest a speci¢c permeability of the
outer mitochondrial membrane without alteration
of the inner mitochondrial membrane.
So far, several competing models exist to explain
exactly how permeabilization of mitochondrial mem-
branes is mediated during apoptosis (see Fig. 2) [88]:
the outer mitochondrial membrane might rupture as
a result of swelling of the mitochondrial matrix. In
one model swelling is postulated to result from open-
ing of a megachannel called the permeability transi-
tion pore (PTP). The adenine nucleotide translocator
(ANT; located in the inner mitochondrial mem-
brane) and the voltage-dependent anion channel
(VDAC, found in the outer mitochondrial mem-
brane) are major components of the PTP which is
proposed to span both the inner and the outer mi-
tochondrial membranes at sites at which the two
membranes are opposed. According to the PTP mod-
el, PTP openers, including the pro-apoptotic Bcl-2
family member Bax, cause permeabilization of the
inner membrane and mitochondrial depolarization
by binding to the ANT [100] (although this is not
supported by the data mentioned above). This pro-
cess allows entry of water and solutes into the matrix
and leads to mitochondrial swelling. Another model
postulates that swelling is due to a defect in mito-
chondrial ATP/ADP exchange as a result of closure
of the VDAC thus leading to hyperpolarization of
the inner mitochondrial membrane and subsequent
matrix swelling. Again such a scenario contradicts
reports stating that the drop in membrane potential,
at least in some cell types, follows the release of
cytochrome c.
Other models do not predict damage of the outer
F8 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37

Fig. 2. Di¡erent models explain the release of cytochrome c from mitochondria during apoptosis. The outer mitochondrial membrane
might rupture as a result of swelling of the mitochondrial matrix. This could be explained with opening of the permeability transition
pore (PTP) or with the closure of the voltage-dependent anion channel (VDAC). Other models relate cytochrome c release to pore
formation allowing the passage of cytochrome c into the cytosol.

mitochondrial membrane but rather the formation of
a pore in this membrane allowing the passage of
cytochrome c (and other mitochondrial proteins)
into the cytosol. Bax is a candidate for the formation
of this pore. Bax oligomers can form large conduc-
tance channels in lipid planar bilayers [88]. Addition
of Bax directly to isolated mitochondria triggers re-
lease of cytochrome c through a mechanism that is
insensitive to PTP blockers and does not involve mi-
tochondrial swelling.
Yet another model involves Bax cooperating with
the VDAC to form a cytochrome c-conducting chan-
nel [101]. Nevertheless, direct evidence for the forma-
tion of such pore structures in mitochondria during
apoptosis is still missing.
An interesting link between death receptor-acti-
vated apical caspases such as caspase-8 and mito-
chondrial cytochrome c release has been established
in the form of the BH3-domain-only protein Bid:
caspase-8 ^ initially activated at the death-inducing
signaling complex (DISC) of cell surface death recep-
tors ^ cleaves the pro-apoptotic Bcl-2 family member
Bid [66,67]. Cleaved Bid (tBid) then binds to Bax
leading to Bax oligomerization and integration into
the outer mitochondrial membrane where it triggers
cytochrome c release [102]. Similarly, tBid binds to
and oligomerizes another pro-apoptotic Bcl-2 homo-
logue, Bak, resulting in cytochrome c release [103].
While studies in bak knockout cells show that tBid
does not require Bak for mitochondrial targeting,
Bak proved necessary for tBid-induced cytochrome
c release. Consequently, bax3/3bak3/3 double
knockout cells are resistant to a wide range of apo-
ptotic stimuli [104].
Several proteins in addition to cytochrome c are
released from mitochondria in cells induced to
undergo apoptosis. Among them is the recently iden-
ti¢ed Smac/Diablo molecule which binds to, and in-
activates, IAPs [105,106]. IAPs inhibit cell death by
binding to procaspases and activated caspases, there-
by blocking their processing and their activity. Smac/
Diablo is released from the mitochondria along with

BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart

 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
cytochrome c during apoptosis and relieves inhibi-
tion of caspase-9 activation by IAP inactivation
[107]. It is also possible that in some cells (type II
cells) Smac/Diablo is required to inactivate an IAP
preventing direct caspase-3 activation by caspase-8.
In this scenario cytochrome c release might not be
relevant for the death process, but rather Smac/Dia-
blo liberation into the cytosol. Smac/Diablo and the
pro-apoptotic Drosophila proteins Reaper, Grim and
Hid seem to function in a similar way (by inhibiting
IAP activity) and a sequence similarity among these
proteins (restricted to their N-terminal 14 amino
acids) has been reported [108] suggesting that
Smac/Diablo and the insect apoptosis-inducing pro-
teins might be structural as well as functional homo-
logues.
Mitochondrial integrity is important not only for
sequestering cytochrome c and Smac/Diablo but also
for other ways to regulate caspase activation and
apoptosis [87]. A fraction of both caspase-9 and cas-
pase-3 has been localized to the mitochondrial inter-
membrane space in some cell types, and caspase-2
has also been reported to reside in mitochondria.
These caspases ^ like cytochrome c ^ can be released
from the mitochondria to the cytosol during apopto-
sis induction. Another protein, AIF (apoptosis-in-
ducing factor), also redistributes from mitochondria
and induces some of the nuclear morphology associ-
ated with apoptosis in a caspase-independent manner
[109]. Genetic inactivation of AIF renders embryonic
stem cells resistant to cell death after serum depriva-
tion and disables PCD during caviation of embryoid
bodies in early mouse morphogenesis [110].
4.3. The death receptor pathway
Recently, a direct mechanistic link between a par-
ticular apoptotic stimulus and activation of the basal
caspase apoptotic machinery has been forged: acti-
vation of a speci¢c group of transmembrane recep-
tors of the tumor necrosis factor (TNF) receptor
superfamily, either by ligand or (experimentally) by
binding an agonistic antibody, can lead to direct ac-
tivation of caspases. The list of TNF receptor family
members is growing and includes TNF-R1 (P55),
TNF-R2 (P75), TNF-R3 (TNF-RP), LT-KR, Ox-
40, CD27, CD28, CD30, CD40, 4-1BB, p75 NGF-
R (low a¤nity nerve growth factor receptor), GIT-R
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F9
[111], Rank, CD95, DR6 [112] and the newly discov-
ered TRAIL receptors TRAIL-R1 (DR4), -R2
(DR5), -R3 (DcR-1) and DcR-2 [113]. Activation
of members of this receptor family triggers a variety
of cellular responses depending on cell type and con-
text, amongst which are (T-cell) activation and stim-
ulation, proliferation, di¡erentiation, survival and
apoptotic cell death [114^116].
Mammalian TNF-R family members are type I
membrane proteins characterized by conserved extra-
cellular cysteine-rich domains. A functional TNF
superfamily receptor is typically a trimeric or multi-
meric complex stabilized by disul¢de bonds,
although some, such as CD95, TNF-R1 and TNF-
R2, also exist in a soluble form generated by proteo-
lytic cleavage [117]. The receptors' ligands comprise
another related family that includes TNF, LT-K
(lymphotoxin-K), CD95 ligand (FasL/CD95L),
TRAIL, OX40L, CD27L, CD30L, CD40L, 4-1BBL
and LT-L. Each of the ligands is synthesized as a
nascent type II membrane-associated protein and
shares a characteristic 150 amino acid region towards
the C-terminus by which each ligand interacts with
its cognate receptor. For the most part, these ligands
exist as trimeric or multimeric membrane-bound pro-
teins that may function to induce receptor aggrega-
tion. However, a few members, such as TNF and
CD95L, are also functional in soluble form. Interest-
ingly, a domain N-terminal to the ligand binding
domain in the extracellular region of TNF-R1,
TNF-R2 and CD95 was recently identi¢ed that
mediated receptor self-association before ligand
binding [118]. This pre-ligand binding assembly do-
main (PLAD) is critical for assembly of functional
receptor complexes on the cell surface. Thus, TNF
receptor family members might function as pre-
formed complexes rather than as individual receptor
subunits that oligomerize after ligand binding.
A detailed discourse on the multiple pleiotropic
cellular and physiological activities provoked by liga-
tion of TNF receptor family members in various cell
types is beyond the scope of this article. We will
con¢ne ourselves to those receptors whose ligation
has been shown to induce apoptosis ^ namely
TNF-R1, CD27 [119], CD30, CD40, LT-LR, CD95,
DR3, DR4, DR5 and DR6 [120]. A subgroup of
these receptors shares a common intracellular pro-
tein^protein interaction domain, the so-called death
F10 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
domain. These receptors are referred to as death re-
ceptors and they include TNF-R1, CD95, DR3,
DR4, DR5, and DR6.
In particular, we shall summarize current knowl-
edge concerning the CD95, TNF-R1 and the TRAIL
receptors and the attendant molecules mediating
their death signal transduction.
4.3.1. CD95/FAS/Apo-1
CD95/Fas/Apo-1, henceforth called CD95, is ex-
pressed in activated lymphocytes as well as in all
other tissues such as the liver, heart and lung. Liga-
tion of CD95, whether by its ligand CD95L or, ex-
perimentally, by agonistic antibody can induce apo-
ptosis in several cell types. CD95L is predominantly
expressed on activated lymphocytes, NK cells, eryth-
roblasts and immune privilege tissues, but also on
certain tumors. The CD95/CD95L apoptotic path-
way also functions to maintain homeostasis in vari-
ous tissues ^ the liver being a particularly well docu-
mented example [121]. However, the biological role
of CD95 is probably best understood in the immune
system, where it is implicated in peripheral clonal
deletion of T-lymphocytes, activation-induced suicide
of mature T-cells, cytotoxic response and induction
of apoptosis in B-cells. Constitutive cell surface
expression of CD95L also seems to contribute to
immunological privilege of certain organs by killing
in¢ltrating lymphocytes and in£ammatory cells
expressing CD95 receptor [122]. Mice carrying
mutations in the genes for CD95 (lpr for lymphopro-
liferation) and CD95L (gld for generalized lympho-
proliferative disease) have been identi¢ed. Mice ho-
mozygous for either of these mutations accumulate
an excess of non-malignant CD3‡ B220‡ CD43 CD83
T-cells in their spleens and lymph nodes and also
su¡er from an autoimmune systemic lupus erythema-
tosus-like condition. This demonstrates that CD95/
CD95L signaling ful¢ls an important function in de-
leting autoreactive lymphocytes and maintaining pe-
ripheral tolerance. Mutations in the human CD95
gene cause a similar lympho-accumulative syndrome
[123]: patients with autoimmune lymphoproliferative
syndrome type 1A have heterozygous CD95 germ-
line mutations and their lymphocytes are resistant
to CD95-induced apoptosis [124].
No identi¢able catalytic motifs are present in the
cytoplasmic domains of the CD95/TNF-R1 recep-
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
tors. Rather, signal transduction is mediated via di-
rect recruitment of, and intermolecular association
with, various downstream signaling e¡ector mole-
cules [51]. In this regard, a key intracellular interac-
tion domain present in the cytoplasmic tail of all
death receptors is the 65 amino acid `death domain'
(DD), a name deriving from its ability to recruit
downstream e¡ectors that can induce apoptosis
[125]. However, the term `death domain' is somewhat
unfortunate, since it implies that cell death is the
generic function of this type of motif. In fact, DDs
are domains that mediate homo- and heterotypic
protein^protein interactions in order to propagate
signals, and they have since been found in signaling
pathways that have no obvious link with cell death
[126].
The DDs in the ligated TNF-R1/CD95 receptors
recruit the C-terminal DD of the cytoplasmic adapter
FADD/MORT-1. At its N-terminus, FADD/MORT
possesses a di¡erent protein binding domain, a
`death e¡ector domain', that mediates interaction
with the N-terminal prodomain of caspase-8
[127,128]. The recruitment of caspase-8 by FADD/
MORT to the activated CD95 receptor generates a
DISC [129] that leads to proteolytic autoactivation
of caspase-8. Caspase-8 then activates other caspases,
including caspase-1 and caspase-3, which then are
presumed to execute the apoptotic dissolution of
the cell [130]. Members of the TNF receptor family
which lack a death domain (e.g. TNF-R2, CD27,
CD30, CD40) are also under certain circumstances
able to induce cell death via alternative mechanisms
[120].
CD95, through recruitment of the DISC, appears
to provide a direct link between external ligand and
the basal e¡ector machinery of apoptosis. However,
it has recently become clear that this direct molecular
cantilever only seems to operate in certain cell types
^ type 1 cells [131]. In other (type 2) cells, CD95
leads to changes in mitochondria that activate down-
stream caspases in a di¡erent way. The amount of
receptor-activated caspase-8 in type 2 cells is much
lower than in type 1 cells [132] and probably insu¤-
cient to induce downstream e¡ector caspase cleavage.
It nevertheless is enough to cleave Bid, a BH3 do-
main-only member of the Bcl-2 family [66,67]. Trun-
cated Bid then translocates to the mitochondria
where it induces cytochrome c release and conse-
 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
quently further caspase activation and ¢nally cell
death.
The DDs of both CD95 and TNF-R1 interact with
the C-terminal DD of a second receptor-associated
protein, designated RIP [133]. The RIP N-terminus
resembles a tyrosine kinase domain which is intrigu-
ing because experimental data implicate a tyrosine
kinase in CD95-mediated signal transduction. Phar-
macologic inhibitors of protein kinases block, in a
concentration-dependent manner, CD95-induced
DNA fragmentation and prolong cell survival [134].
The DD of RIP also binds to the C-terminal DD of
another `death adapter protein', RAIDD (for RIP-
associated ICH-1/Ced-3 homologous protein with a
death domain). At its N-terminus, RAIDD is homol-
ogous to, and oligomerizes with, the prodomain of
caspase-2 (Ich-1). Thus, caspase-2 can be recruited to
the CD95 receptor through sequential interactions of
RAIDD, RIP, FADD and CD95 [115]. A close rel-
ative of RAIDD is CRADD, which also interacts
with both RIP and caspase-2 [135]. RIP is required
for TNF-induced NF-UB activation. Cleavage of RIP
by caspase-8 results in the blockage of NF-UB-medi-
ated anti-apoptotic signals [136].
Recently, another CD95 binding protein, Daxx,
has been described [137]. Daxx also binds to the
CD95 death domain but lacks a death domain of
its own. Overexpression of Daxx activates Jun
N-terminal kinase (JNK) and potentiates CD95-in-
duced apoptosis. On this basis, it has been proposed
that CD95 engages two independent pathways that
induce cell death: one pathway via FADD/caspase-8/
2 and the other via Daxx/JNK activation. Interest-
ingly, Daxx is a nuclear protein that interacts and
colocalizes with the tumor-suppressive promyelocytic
leukemia protein PML in nuclear bodies [138]. Re-
porter gene assays show that DAXX is able to re-
press basal transcription; SUMO-1-modi¢ed PML
sequesters DAXX to the nuclear bodies and inhibits
Daxx-mediated transcriptional repression. How pre-
cisely CD95 activation acts on Daxx localization,
and its in£uence on transcription, is presently un-
clear. Strangely enough, rather than showing de-
creased apoptosis, inactivation of Daxx results in
extensive apoptosis and embryonic lethality in mice
[139].
Among the proteins which have been shown to
bind to the cytosolic domain of the CD95 receptor
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F11
is the Fas-interacting serine/threonine kinase/homeo-
domain-interacting protein kinase FIST/HIPK3
[140]. FIST not only binds to CD95 but also inter-
acts with FADD in a trimolecular complex com-
posed of CD95, FADD and FIST. FIST kinase in-
duces FADD phosphorylation and inhibits CD95-
mediated JNK kinase activation. It is localized
both in the cytoplasm and in the nucleus and is ca-
pable of binding to Daxx in a kinase activity-depen-
dent manner.
In addition to activation of caspases and JNKs,
both CD95 and TNF-R1 trigger other signaling ef-
fectors. CD95-generated apoptotic signals activate
acidic sphingomyelinase causing accumulation of cer-
amide [141] which is observed in both CD95- and
TNF-R1-induced apoptosis. Whether ceramide pro-
duction is a major determinant of the apoptotic de-
cision is still a matter of debate.
Naturally occurring inhibitors of the CD95/TNF-
R1 death signaling pathways exist in the guise of the
FLIPs (Fas-associated death domain-like ICE inhib-
itory proteins) which interfere with recruitment of
caspases to the CD95/TNF-R1 signaling complexes.
A number of viruses encode FLIPs as part of their
strategy for manipulating host cell suicide and viabil-
ity. For example, the Q-herpesviruses encode FLIPs
that comprise two death e¡ector domains which in-
teract with FADD/MORT and inhibit its recruitment
and activation of caspase-8 [142]. Recently, a cellular
homologue of v-FLIP was identi¢ed by di¡erent
groups [143^150]. c-FLIP is structurally similar to
caspase-8 since it contains two death e¡ector do-
mains and an inactive caspase-like domain lacking
the conserved functional cysteine residue. c-FLIP is
expressed in two isoforms (long and short form),
both of which are recruited to the CD95 DISC in a
stimulation-dependent fashion. c-FLIP blocks cas-
pase-8 activation at the DISC and thereby inhibits
CD95-mediated apoptosis [151]. During this process,
both caspase-8 and c-FLIP undergo cleavage be-
tween the p18 and p10 subunits, generating two sta-
ble intermediates of 43 kDa that stay bound to the
DISC.
B- and T-cells downregulate c-FLIP upon activa-
tion in vitro [152], providing a possible explanation
for the observation that resting peripheral T-cells are
resistant to CD95-induced apoptosis and become
susceptible only after their activation. By inhibiting
F12 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
death receptor-mediated cell death, c-FLIP has been
identi¢ed as a tumor progression factor in mouse
models [153,154]. Several groups found a pro-apo-
ptotic function of c-FLIP in transient overexpression
studies [146], the physiological relevance of which is
presently unclear [151].
Another way to inhibit death ligand-induced apo-
ptosis is to quench the signal via decoy receptors. A
soluble CD95 decoy receptor (DcR3) has been dis-
covered that binds to CD95L and inhibits CD95L-
induced apoptosis [155,156]. The physiological im-
portance of such signal inhibition is underlined by
the ¢nding that the DcR3 gene was ampli¢ed in
about half of the primary lung and colon tumors
studied.
Knowledge of the downstream e¡ectors involved
in CD95 death signaling has facilitated analysis of
the role of CD95 in vivo. As discussed above, mice
with inactivating mutations in the genes for either
CD95 (lpr) or CD95L (gld) exhibit generalized lym-
phoproliferative disease. The cowpoxvirus caspase
inhibitor CrmA, which e¤ciently blocks caspase-8
(as well as other caspases such as caspase-1), has
been expressed transgenically in peripheral T-lym-
phocytes via the CD2 promoter. Such CD2-crmA
transgenic mice exhibit resistance to CD95-induced
apoptosis equivalent to that seen in lpr mice [157]
although neither Q-radiation- nor corticosteroid-in-
duced cell death is suppressed. However, in contrast
to lpr mice, CD2-crmA transgenic mice develop nei-
ther T-cell hyperplasia nor serum autoantibodies, im-
plying that the lpr phenotype is not merely due to
failure of CD95 to trigger caspase-dependent T-cell
apoptosis. Expression of a dominant negative mutant
of FADD in T-lymphocytes also severely repressed
CD95 killing yet failed to cause accumulation of pe-
ripheral T-cells as seen in lpr and gld mice [158,159].
Mice with a deletion in the FADD gene die at day
11.5 of embryogenesis; their phenotype suggests that
FADD is essential for embryo development and sig-
naling from some (but not all) inducers of apoptosis
[160]. Interestingly, inactivation of FADD by expres-
sion of a FADD dominant negative molecule or by
gene targeting leads to impairment of activation-in-
duced T-cell proliferation [158,159,161,162].
CD95 is also interesting in another aspect of tu-
mor therapy: several anticancer drugs have been
shown to sensitize certain cell types to apoptosis by
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
upregulating CD95 or CD95L [163,164] although the
generality of this concept has been questioned
[165,166].
4.3.2. TNF receptors
TNF is well recognized as a cytokine produced by
activated T-cells and macrophages that orchestrates
aspects of the host in£ammatory response. It does so
by in£uencing the proliferation, di¡erentiation and
apoptosis of cells involved in in£ammation. TNF
(together with the lymphotoxin LT) is the ligand
for two receptors ^ TNF-R1 and TNF-R2. TNF-
R1 alone appears to be able to mediate most, if
not all, of the biological responses engendered by
TNF, although TNF-R2 may provide an auxiliary
function in cooperating in the binding of TNF to
TNF-R1 [167]. Genetic deletions of both receptors
have demonstrated the di¡erences in biological func-
tionality of TNF-R1 and TNF-R2 in vivo [168^170].
Although both act to potentiate in£ammation/host
defense and share the common ability to activate
the pleiotropic transcription factor NF-UB [171],
TNF-R1 alone can clearly trigger apoptosis whereas
TNF-R2 mainly seems to promote cell survival,
although it was shown to kill certain cells when over-
expressed [120]. However, substantial evidence indi-
cates that TNF-R1 can also promote cell survival
under certain circumstances, although this anti-apo-
ptotic activity, unlike activation of the caspase cas-
cade, appears to be indirect and require de novo syn-
thesis of survival proteins.
TNF-R1 signaling, like CD95, is able to activate
the proteolytic caspase cascade by recruiting caspase-
8 via FADD/MORT. Although the FADD/MORT
adapter molecule does not bind directly to TNF-R1,
it is recruited to the activated receptor via an inter-
mediary cytoplasmic DD adapter called TRADD
(TNF-R-associated death domain). TRADD also
binds RIP, thereby linking TNF-R1 to caspase-2 ac-
tivation via RAIDD and CRADD. Both TNF-R1
and TNF-R2 recruit another class of signaling adapt-
er molecules, called TRAFs (TNFR-associated fac-
tors) of which six are currently identi¢ed [172]. Cer-
tain of the TRAFs mediate activation of JNK or
NF-UB [173], the latter by interaction with the down-
stream signaling kinase NIK. NIK, in turn, activates
the IUB kinases which phosphorylate and inactivate
IUB, the endogenous cellular inhibitor of NF-UB
 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
[174]. Substantial evidence supports the notion that
NF-UB can act as a powerful suppressor of apoptosis
providing an explanation for how TRAFs might ex-
ert their anti-apoptotic activity. Based on their NF-
UB-dependent expression and anti-apoptotic func-
tion, the cIAPs, TRAF-1 and TRAF-2 as well as
A20 have been proposed to play some role in NF-
UB-mediated prevention of apoptosis [175,176].
TRAFs are held in abeyance in the cytoplasm
through their association in oligomeric complexes
with I-TRAF [177]. All TRAFs share a conserved
V230 amino acid `TRAF' domain which mediates
their homo- or hetero-oligomerization with other
TRAFs, their interaction with the cytoplasmic tails
of members of the TNF-R superfamily, and interac-
tions with downstream signal transducers [173]. In
addition to the TRAF domain, most of the TRAFs
also contain an N-terminal ring ¢nger plus several
zinc ¢nger structures which appear to be important
for their various e¡ector functions.
TRAF proteins are signal transduction adapter
proteins. TRAF-2, -5, and -6 have been shown to
be mediators of both NF-UB activation and SAPK/
JNK activation [178]. The activation processes in-
volve successions of protein^protein interactions
and phosphorylation of protein kinases. The pheno-
types of Traf-2 knockout mice and transgenic mice
expressing a dominant negative mutant of TRAF-2
indicate that TRAF2 is important for regulation of
lymphocyte proliferation and survival [179,180].
They are also compromised for JNK activation,
although NF-UB activation is una¡ected.
TRAF-2, TRAF-5, and TRAF-6 interact with the
downstream kinase NF-UB-inducing kinase (Nik),
which in turn interacts with the kinases within the
IUB kinase complex [178]. In addition, the death do-
main kinase RIP and the serine/threonine kinase
IRAK have also been reported to interact with
TRAF proteins and mediate NF-UB activation. On
the other hand, apoptosis signal-regulating kinase
ASK-1, a TRAF-interacting kinase, was recently
demonstrated to be a downstream target of TRAF-
2, TRAF-5, and TRAF-6 in the JNK signaling path-
way.
One particularly intriguing potential target of the
TRAFs are the IAP proteins. Originally discovered
in baculovirus as suppressors of host cell apoptosis,
the iap [181] genes now comprise a large evolutionar-
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F13
ily conserved family of viral and cellular genes. IAP
proteins are characterized by the presence of so-
called BIR (baculovirus IAP repeats) motifs. These
65 amino acid repeats are typically found in the
N-terminal region of the IAPs and mediate various
types of protein^protein interactions. Certain IAPs
also contain a C-terminal ring ¢nger which is pre-
sumed to mediate other speci¢c protein^protein in-
teractions and acts as a ubiquitin ligase, promoting
the degradation of the X-linked IAP (XIAP) [182].
Like their viral homologues, several cellular IAPs are
implicated in suppression of apoptosis. The gene en-
coding one member of the human IAP family, NAIP
(neuronal apoptosis inhibitory protein), is partially
deleted in individuals with spinal muscular atrophy,
a disease involving apoptosis of motor neurons [183].
Moreover, human cIAP-1 and cIAP-2 have both
been biochemically puri¢ed as part of the TNF-R2-
TRAF signaling complex [184], an interaction involv-
ing the IAPs' BIR domains and the N-terminal do-
main of TRAF-2. Although interference with TNF
signaling could partly explain cellular IAP anti-apo-
ptotic activity, at least one other potent anti-apopto-
tic human IAP homologue, hILP, exhibits no inter-
action with any tested TRAF protein. Nonetheless, it
is possible that some cellular IAPs act to modulate
TRAF activation of the anti-apoptotic NF-UB tran-
scription factor.
Another member of the IAP family, survivin, has
been found to be overexpressed in tumor cells while
undetectable in terminally di¡erentiated adult tissues
[185]. These observations potentially place survivin in
the same class of cell death-inhibiting oncogenes as
bcl-2.
IAPs are conserved during evolution and have
been identi¢ed in Drosophila, C. elegans and in yeast.
Nevertheless, while the Drosophila caspase inhibitor
DIAP-1 is essential for cell survival [186,187] as
mammalian IAPs are inhibited by Smac/Diablo
(like cytochrome c released from mitochondria dur-
ing apoptosis [105,106]), the C. elegans and yeast
IAPs do not seem to play a role in the inhibition
of cell death but are rather involved in the regulation
of cytokinesis and cell division, respectively
[188,189]. Such a role has also been identi¢ed for
the mammalian survivin protein, in addition to its
anti-apoptotic function [190,191].
A possible model to explain the contradictory ap-
F14 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
optotic and anti-apoptotic outcomes of TNF recep-
tor signaling emerges from the cloning of another
TRAF-interacting protein, TRIP [173]. TRIP con-
tains a ring ¢nger motif and an extended coiled-coil
domain and it associates with TNF receptor family
members through its interaction with TRAF pro-
teins. When so associated, TRIP inhibits TRAF-
mediated activation of the apoptosis suppressor
NF-UB. Thus, TRAF interactions with cIAPs would
suppress apoptosis whilst interactions with TRIP
would promote it. Which outcome of TNF signaling
prevails may depend upon the availability of cIAPs
and TRIP and their relative a¤nities for whichever
TRAFs are available in any particular instance.
In another report the authors showed that in the
presence of the serine/threonine kinase RIP (required
for NF-UB activation by TNF-R1), TNF-R2 triggers
cell death in T-cells whereas in the absence of RIP,
TNF-R2 activates NF-UB [192]. RIP is induced dur-
ing interleukin (IL)-2-driven T-cell proliferation, and
its inhibition reduces susceptibility to TNF-depen-
dent apoptosis.
4.3.3. TRAIL receptors
Recently, an interesting subfamily of TNF recep-
tors has been identi¢ed ^ the TRAIL receptors
[113,193,194]. TRAIL (TNF-related apoptosis-induc-
ing ligand), also called apo-2L, is a broadly ex-
pressed TNF-related ligand which appears not to
bind either CD95 or TNF-R1. Like all members of
the TNF/CD95L family, TRAIL is synthesized as a
membrane-bound proprotein which can be cleaved to
generate a soluble ligand. Many human tumor cells
and tumor cell lines are sensitive to induction of ap-
optosis by cell surface or soluble TRAIL [195]. How-
ever, normal cells, such as freshly isolated mouse
thymocytes or primary B- or T-cells, are insensitive.
Such a cell type-dependent response might be con-
strued as indicative of a restricted receptor distribu-
tion. However, the ¢rst two identi¢ed TRAIL bind-
ing receptors (DR4/TRAIL-R1 and DR5/TRICK-2/
TRAIL-R2/KILLER/DR5) are expressed in most
human tissues and some tumor cell lines. TRAIL
induces apoptosis through these two death domain-
containing receptors requiring FADD and caspase-8,
just like CD95-mediated cell killing [196]. Neverthe-
less, three other TRAIL receptors have since been
identi¢ed. Two of these receptors, decoy receptor 1
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
(DcR1/TRID/LIT/TRAIL-R3) and decoy receptor 2
(DcR2/TRUNDD), lack any functional death do-
main and are therefore unable to transduce a death
signal (as is the third receptor, osteoprotegerin, a
secreted protein). They can therefore act as `decoy
receptors' by abstracting TRAIL ligand from pro-
ductive interactions with DR4 or DR5 and their
presence could explain why some cells are preferen-
tially killed by TRAIL and others not. Indeed, DcR1
and DcR2 are expressed in many tissues although
not in most cancer lines examined. However,
although apoptosis depends on the expression of
one or both of the death domain-containing recep-
tors DR4 and/or DR5, resistance to TRAIL-induced
apoptosis does not correlate with the expression of
the `decoy' receptors. One possibility is that rather
than binding to all its receptors with equivalent high
a¤nities, TRAIL a¤nity di¡erences exist at physio-
logical temperature, and the cell death-inducing DR5
receptor seems to be the highest a¤nity receptor
[197]. So the ability of TRAIL to speci¢cally kill
tumor cells still awaits a satisfying explanation.
Not much is known about TRAIL's normal bio-
logical function. A TRAIL-dependent mechanism of
monocyte-induced cell cytotoxicity has been reported
suggesting that TRAIL might be an important e¡ec-
tor molecule in antitumor activity in vivo [198].
Preclinical studies with mice and non-human pri-
mates have shown that indeed TRAIL induced the in
vivo regression of tumors ^ in contrast to TNF or
CD95 ligand/CD95 antibody ^ without severe side
e¡ects [199]. In particular, a combined treatment of
tumor cells with either chemotherapeutics or ionizing
radiation gave promising results [200,201]. Neverthe-
less, a recent publication reported that human pri-
mary hepatocytes (in contrast to mouse hepatocytes)
are also e¤ciently killed by TRAIL [202,203], a ¢nd-
ing that has to be taken into account for future clin-
ical trials involving TRAIL. Human astrocytes seem
to represent another untransformed cell type sensi-
tive to TRAIL-mediated killing [199].
5. Cell death regulators
5.1. Oncoprotein-induced cell death
A number of oncoproteins induce apoptosis when
 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
overexpressed in cells [204]. The best characterized
examples are the transcription factor c-Myc [205]
and the adenovirus protein E1A [206], but the list
also includes c-Jun [207] and c-Fos [208], both com-
ponents of the AP-1 transcriptional complex, as well
as components of the G1 cell cycle progression ma-
chinery such as E2F1 and cyclin E (while induction
of apoptosis seems to represent the general cellular
response upon oncogene overexpression, in certain
settings the reduction of c-Myc [209], Jun or Fos
[210] expression via an unknown mechanism leads
to apoptosis). Because all such oncoproteins promote
cell proliferation, their pro-apoptotic activity was
often interpreted as arising from some kind of abor-
tive or failed attempt at going through the cell cycle
^ a mitotic or S phase catastrophe. However, with
the elucidation of its underlying mechanisms it be-
came clear that apoptosis is not merely a corrupted
cell cycle. An alternative variant of the same idea is
that oncogene-induced apoptosis arises through a
con£ict of growth signals: oncogenes activate apo-
ptosis if their proliferative action is blocked in
some way or if the cell's proliferative machinery is
incompletely activated or coordinated. However, the
idea of a con£ict of signals, whilst intuitively plausi-
ble, does not o¡er any explanation as to the molec-
ular nature of such a con£ict, how it arises or is
de¢ned, or why any con£ict (were it to exist) triggers
the machinery of apoptosis.
The current view of Myc-induced apoptosis is ex-
pressed in the `dual signal' hypothesis [211]. Accord-
ing to this hypothesis Myc promotes both pathways
at the same time ^ proliferation and apoptosis. The
apoptotic pathway is suppressed as long as appropri-
ate survival factors deliver anti-apoptotic signals.
Such a scenario would ¢t with a general model of
survival/cell death regulation according to which the
`default' fate of a cell would be cell death unless
suppressed by anti-apoptotic cytokine signaling
[212]. Coupling of the `contradictory' pathways of
cell proliferation and cell death downstream of pro-
teins like c-Myc incorporates a potent safety mecha-
nism for the suppression of carcinogenesis: any le-
sion that activates the mitogenic pathway will prove
lethal should the a¡ected cell and its progeny out-
grow the paracrine environment enabling their sur-
vival [213].
Since the cell death-promoting activity of onco-
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F15
genes might hinder expansion of potentially malig-
nant cells, proteins such as Myc or E2F may, in
certain circumstances, act as tumor suppressors.
For example, E2F-1 knockout mice develop dyspla-
sias and lymphocytic hyperplasias [214,215], in part
because of insu¤cient apoptosis (nevertheless, loss of
E2F-1 reduces tumorigenesis and extends the lifespan
of Rb-1(+/3) heterozygous knockout mice [216],
demonstrating that E2F-1 also positively regulates
cell cycle progression).
Mutagenesis studies show that regions of the
c-Myc protein necessary for apoptosis induction over-
lap with regions important for co-transformation
and include the N-terminal transactivation domain
and the C-terminal bHLH-LZ region (basic helix-
loop-helix domain with a leucine zipper) involved
in sequence-speci¢c DNA binding and dimerization
with Max [211]. The heterologous protein Max (re-
quired for Myc as a binding partner for DNA bind-
ing) is absolutely required for induction of apoptosis
by c-Myc in ¢broblasts [217] while Mad-1 overex-
pression (antagonizing Myc functions by repressing
gene transcription) inhibits proliferation and apopto-
sis [218]. These data strongly argue that c-Myc in-
duces apoptosis through its action as a transcription
factor ^ presumably by modulation of appropriate
target genes.
Several candidate Myc-regulated genes have been
identi¢ed [219,220]. Two genes suggested as Myc tar-
gets, based on the pattern of expression, are orni-
thine decarboxylase (ODC) and cdc25A. ODC is a
rate-limiting enzyme in polyamine biosynthesis, nec-
essary for DNA synthesis, while Cdc25A encodes a
tyrosine phosphatase involved in activation of the
key inducers of mitosis, the cyclin-dependent kinase
(CDK) complexes. Both candidate Myc targets,
ODC and cdc25A, when overexpressed, induce apo-
ptosis in cells lacking survival factors (as Myc over-
expression does). It therefore seems possible that the
cell proliferation and cell death pathways bifurcate
`downstream' of c-Myc. In addition the TNF-R-as-
sociated protein TRAP-1 [221] and the Bcl-2 family
member Bax [222] have been revealed as further Myc
targets possibly involved in Myc-induced apoptosis.
The ¢nding that c-Myc (and E1A) activates P53
via p19ARF , a protein encoded by the alternate read-
ing frame of the p16 tumor suppressor gene, has
provided another possible mechanism linking c-Myc
F16 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
with the induction of apoptosis [223,224]. In this
context it is interesting to note that E2F-1 seems to
induce apoptosis by upregulation of the P53 homo-
logue P73 [225,226].
A novel Myc-interacting protein, Bin-1, has been
shown to induce apoptosis and di¡erentiation in neu-
roblastoma [227] but the functional relation to Myc-
induced apoptosis is unclear.
Recently, it was revealed that c-Myc-induced apo-
ptosis requires interaction on the cell surface between
CD95 and CD95L [228]. With this study the two
previously independent apoptosis pathways (Myc
and CD95/CD95L) have been interconnected. Dur-
ing Myc-induced apoptosis in serum-deprived ¢bro-
blasts c-Myc acts downstream of the CD95 receptor
by sensitizing cells to the CD95 death signal,
although in general c-Myc may also act upstream
of CD95/CD95L by inducing expression of their cog-
nate genes.
Expression of c-Myc sensitizes cells to a wide
range of pro-apoptotic stimuli. The best hint towards
the mechanism underlying Myc-mediated sensitiza-
tion comes from studies showing that this pro-apo-
ptotic e¡ect is mediated through release of mitochon-
drial holocytochrome c into the cytosol [229].
Cytochrome c release is caspase-independent and is
blocked by the survival factor insulin-like growth
factor (IGF)-1. While neither P53 nor CD95/Fas sig-
naling was required for Myc-induced cytochrome
c release, a dominant negative version of the adapter
protein FADD (inhibiting CD95 signaling) blocked
caspase activation subsequent to cytochrome c re-
lease. The emerging model suggests that c-Myc pro-
motes apoptosis by causing the release of cytochrome
c, but the ability of cytochrome c to promote apo-
ptosis is critically dependent upon other signals such
as CD95 activation. Myc's cytochrome c-releasing
activity might be mediated by upregulation of the
pro-apoptotic Bcl-2 family member Bax which has
been suggested as a transcriptional target and medi-
ator of c-Myc-induced apoptosis [222].
The fact that IGF-1 inhibits c-Myc-induced cyto-
chrome c release but not c-Myc-dependent prolifer-
ation represents additional evidence that the mito-
genic and apoptotic pathways downstream of Myc
are distinct and separate.
While the importance of Myc-induced apoptosis is
evident in an unphysiological situation like tumori-
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
genesis (many tumors show deregulated Myc expres-
sion and necessarily need to counteract Myc's pro-
death function), it has been di¤cult to prove phys-
iological relevance of Myc-induced apoptosis. One
reason for that is the di¤culty to delete Myc func-
tion (which is needed for proliferation) and thereby
showing impairment of certain apoptotic processes.
The demonstrated dependence of Myc on CD95 sig-
naling and vice versa for cell killing raises the possi-
bility that every physiological cell death requiring
CD95/CD95L also depends on Myc function. It
has also been shown that c-Myc (and E1A) sensitizes
target cells for the cytotoxic action of activated NK
cells [230] thereby possibly directing NK cytotoxicity
towards virus-infected and cancer cells.
The precise consequences of Myc overexpression
in vivo seem to depend on the particular type of
tissue and its potency to suppress apoptosis via sur-
vival signals. For example, transgenic expression of a
regulatable c-Myc protein in suprabasal keratino-
cytes of the mouse epidermis results in premalignant
papillomatous skin lesions accompanied by angio-
genesis [231]. Apparently, ectopic Myc activation in
skin causes proliferation with no detectable apopto-
sis, although Myc potently triggers apoptosis in vitro
in isolated serum-deprived keratinocytes from the
same transgenic animals. Myc-induced apoptosis in
intact skin might be suppressed by the presence of
excess survival signals such as cytokines or extracel-
lular matrix attachments. The dominant loss of
c-Myc-activated keratinocytes through shedding
may account for the rarity of malignant progression
of actinic keratosis (although c-Myc alone is su¤-
cient to induce premalignant skin lesions).
In contrast to skin, the predominant e¡ect of Myc
activation in the L-cells of transgenic murine pan-
creas is apoptosis [232] indicating that c-Myc would
be unlikely to initiate a L-cell tumor in the absence of
an anti-apoptotic lesion.
5.2. The Bcl-2 protein family
5.2.1. The Bcl-2 protein
The proto-oncogene bcl-2 was ¢rst discovered as
the target gene present at the translocation site of the
t(14;18) chromosomal translocation breakpoint in
the tumor cells of approximately 80% of patients
with human follicular B-cell lymphoma [233]. The
 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
translocation places the bcl-2 gene under the aegis of
the 5P immunoglobulin heavy chain gene enhancer
(EW, chromosome 14), an element highly transcrip-
tionally active in B-lymphoid cells. Functional stud-
ies of the e¡ects of deregulated bcl-2 on lymphocytes
in culture indicated that bcl-2 exerted a novel type of
oncogenic function ^ rather than promoting cell pro-
liferation or inhibiting di¡erentiation, it suppressed
lymphocyte apoptosis [234].
The anti-apoptotic activity of Bcl-2 explains the
marked oncogenic synergy observed between Bcl-2
and c-Myc. Co-expression of both c-Myc and Bcl-2
induces very rapid genesis of lymphoma [235]. Sev-
eral in vitro studies con¢rm that this synergy arises
because Bcl-2 e¡ectively suppresses c-Myc-induced
apoptosis without signi¢cantly a¡ecting the ability
of c-Myc to drive uncontrolled proliferation [236^
238].
Bcl-2 is expressed in a wide variety of fetal tissues
but in the adult tends to be more restricted to cells
that are rapidly dividing and di¡erentiating.
Bcl-2 knockout mice appear almost normal at
birth but later in life develop hair hypopigmentation
(due to death of melanocytes), distortion of their
small intestines and polycystic kidney disease [239].
Kidneys from Bcl-2-de¢cient mice are small and con-
tain fewer nephrons than those of wild-type animals,
their immune systems exhibit depletion of B- and
T-cells due to apoptosis and this leads eventually to
massive involution of spleen and thymus. In addi-
tion, Bcl-2 knockout mice exhibit marked postnatal
degeneration of motor neurons, sensory and sympa-
thetic neurons. Nevertheless, the largely normal pro-
gression of Bcl-2-de¢cient embryos through develop-
ment (despite the critical nature of apoptosis in
development) attests to a signi¢cant functional re-
dundancy that mitigates the e¡ects of loss of Bcl-2.
5.2.2. Other members of the Bcl-2 family
Bcl-2 is structurally and functionally conserved
throughout metazoan evolution and it is the proto-
type of an extended family of related viral and mam-
malian proteins. This family can be divided into in-
hibitors (Bcl-2, Bcl-xL , Bcl-w, B£-1, Brag-1, Mcl-1,
A1, E1B19K, LMW5-HL and EBV BHRF1) and
promoters (Bax, Bak, Bcl-xS , Bad, Bid, Bik, Hrk,
Bim and Bok) of apoptosis [240,241]. Many members
of the Bcl-2 family share four conserved domains
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F17
(BH1^BH4). BH1, BH2 and BH4, together with
the carboxy-terminal hydrophobic transmembrane
region (membrane anchor) found in most family
members, are important for functional activity. The
BH3 domain seems to be dispensable for the death-
suppressing function of anti-apoptotic Bcl-2 homo-
logues although its presence is essential for cytotoxic
activity in the pro-apoptotic family members [242].
5.2.2.1. Bcl-x. Bcl-xL is the closest mammalian
relative of Bcl-2 and it too acts as a death suppressor
[243]. The 2.7 kb bcl-x transcript encodes a protein
with 44% sequence identity to human and mouse bcl-
2. However, unlike bcl-2, the bcl-x gene encodes two
polypeptides arising from alternative splicing. The
longer mRNA encodes a death-suppressing function-
al Bcl-2 homologue (Bcl-xL ) comprising 233 amino
acids with an apparent molecular weight of 28 kDa.
Bcl-xL contains all four BH domains (BH1^BH4)
found in Bcl-2. The shorter mRNA encodes the
170 amino acid 18 kDa Bcl-xS protein. Compared
to Bcl-xL , Bcl-xS lacks a 63 amino acid stretch which
includes the BH1 and BH2 domains and it may act
as a dominant interfering regulator of either Bcl-xL
or Bcl-2 or both.
Bcl-x is widely expressed and is particularly high in
brain, kidney and adult thymus. Its expression pat-
tern in the di¡erent subsets of T-cells is clearly di¡er-
ent from that of Bcl-2, whose expression is highest in
CD43 CD83 (DN) T-cells, is downregulated in
CD4‡ CD8‡ (DP) thymocytes and rises again in
CD4‡ or CD8‡ (SP) T-cells. Bcl-xL is transcription-
ally activated by NF-UB [244] or by STAT 5 [245].
Transgenic deregulation of Bcl-xL in the lymphoid
lineage has consequences very similar to those of Bcl-
2 overexpression [246,247]: lymphocytes are pro-
tected to varying degrees from killing by dexametha-
sone, Q-irradiation, ionomycin and CD3 ligation but
clonal deletion of T-cells is una¡ected. Moreover,
Bcl-xL will rescue mature T-cells in Bcl-2-de¢cient
mice. The similar functionality of Bcl-2 and Bcl-xL
and the fact that both heterodimerize with pro-apo-
ptotic Bcl-2 family members such as Bax and Bak all
argue that Bcl-xL and Bcl-2 function in a common
pathway to antagonize PCD. However, their di¡er-
ent patterns of expression indicate that their physio-
logical roles are distinct. This is most dramatically
evidenced by the fact that Bcl-xL -de¢cient mice, un-
F18 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
like those lacking Bcl-2, exhibit embryonic lethality
at around day E13 which is accompanied by massive
apoptosis in the brain and hematopoietic tissues
[248].
5.2.2.2. Bax. Bax is a prototypic member of the
Bcl-2 family that accelerates or activates apoptosis
rather than suppressing it. Both Bax and its close
relative Bak possess BH1, BH2 and BH3 regions
but their BH4 is poorly preserved. Bax and Bak cy-
totoxic activity is dependent upon a small region of
each protein that comprises the BH3 domain. The
same BH3 region mediates dimerization between
Bax/Bak and Bcl-2, Bcl-xL , E1B 19K, Bid or itself
[249]. Bax accelerates apoptosis in prolymphocytic
FL5.12 cells upon IL-3 withdrawal [241] and also
counters the death-suppressive activity of Bcl-2: the
higher the expression levels of Bax compared to Bcl-
2, the greater the suppression of Bcl-2 function. On
this basis, a `rheostat' model for Bcl-2/Bax function
has been proposed [250] in which the propensity to
undergo PCD depends upon the relative ratios of
Bax/Bax homodimers, Bcl-2/Bax heterodimers and
Bcl-2/Bcl-2 homodimers: an excess of Bax homo-
dimers promotes cell death, whereas Bax complexed
with Bcl-2 favors survival. It is still not clear, how-
ever, whether Bax promotes PCD, or whether it
merely suppresses the protective e¡ect of Bcl-2, or
whether Bax and Bcl-2 regulate cell viability through
interaction with discrete downstream e¡ectors [251].
Nevertheless, some reports have shown that Bcl-2
and Bcl-x can inhibit apoptosis and Bax can promote
apoptosis by a heterodimerization-independent
mechanism [252^254] and that Bak can accelerate
chemotherapy-induced cell death independently of
its heterodimerization with Bcl-xL and Bcl-2 [255].
Bax is expressed in a wide variety of tissues includ-
ing lymphoid organs, lung, stomach and kidney.
Transgenic expression of Bax in T-cells of mice con-
¢rms the pro-apoptotic activity of Bax [256]: a¡ected
cells exhibit accelerated apoptosis in response to
Q-radiation, dexamethasone and etoposide. When
T-cell-targeted bax transgenic mice are crossed with
their bcl-2 transgenic counterparts, the ratio of Bax
to Bcl-2 present in primary T-cells determines net
sensitivity to apoptotic stimuli, as predicted by the
`rheostat' model.
In vitro and transgenic data con¢rm an important
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
role for Bax in the promotion of apoptosis. Addition
of Bax to isolated mitochondria induces cytochrome
c release through a mechanism that is suppressible by
co-addition of Bcl-xL [257]. In vivo it has also been
shown that Q-radiation induces upregulation of Bax
protein with subsequent apoptosis in radiation-sensi-
tive mouse cells. Such DNA damage-induced expres-
sion of Bax is, in part, mediated by P53 for which a
consensus binding site exists in the human [258] and
in the mouse bax gene (although it was shown that
the putative P53 binding sites were not occupied by
protein in vivo in primary murine thymocytes either
before or after induction of P53 [259]). Thus, for
example, neurons from bax knockout mice show
much reduced apoptosis following DNA damage or
excitotoxin exposure both of which activate P53 re-
sponses [260]. Moreover, Bax de¢ciency fosters drug
resistance as well as oncogenic transformation by
attenuating P53-dependent apoptosis [261]. Indeed,
in a choroid plexus epithelial tumor model induced
by transgenic expression of a truncated SV40 large
T-oncoprotein that lacks the ability to inactivate
P53, absence of Bax greatly diminishes the ability
of P53 to retard tumor growth [262]. Nevertheless,
Bax cannot be the sole apoptotic e¡ector of P53
because thymocytes, and many other cell types,
from Bax knockout mice behave normally with re-
spect to P53-dependent DNA damage responses
[241]. Moreover, transgenic expression of Bax is un-
able to substitute for the absence of P53 in thymo-
cytes exposed to genotoxins [256].
bax knockout mice appear to develop normally,
indicating, as with bcl-2, a su¤cient amount of func-
tional redundancy conferred by other cellular pro-
teins (also bak3=3 mice were found to be develop-
mentally normal while bax3=3 bak3=3 mice display
multiple developmental defects indicating overlap-
ping roles for Bax and Bak in the regulation of ap-
optosis during mammalian development [263]).
Nonetheless, Bax-de¢cient mice demonstrate a few
interesting phenotypes [264]: bax null mice display
thymocyte and B-cell hyperplasia ^ a role perfectly
consistent with the idea of Bax being a positive ef-
fector of apoptosis. Likewise, neurons from bax
knockout mice exhibit marked resistance towards ap-
optosis induced by trophic factor deprivation [265] as
well as insensitivity to toxins. Surprisingly, male bax
knockout mice become sterile due to a block in
 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
sperm production. In this case the pathology linked
to loss of Bax seems to involve rather induction of
excess and ectopic apoptosis. Another interpretation
is that Bax normally mediates elimination of certain
cells during development which, when retained in
bax3=3 mice, lead to inappropriate cell death during
spermatogenesis, perhaps by a¡ecting the availability
of trophic signals.
5.2.2.3. Bad and other BH3 domain only Bcl-2 fam-
ily members. Egl-1 of C. elegans and at least seven
mammalian proteins (Bad, Bik, Blk, Hrk, Bid, Bim,
Noxa) share only the short (nine to 16 residue) BH3
domain with the Bcl-2 family. This domain allows
them to bind to the pro-survival Bcl-2-like molecules
and neutralize their function.
Bim for example was shown to be required for
hematopoietic homeostasis and as a barrier to auto-
immunity. Moreover, particular death stimuli appear
to activate apoptosis through distinct BH3-only pro-
teins [266].
Bad was originally identi¢ed by yeast two-hybrid
screening and expression cloning as a 204 residue
(22.1 kDa) protein that binds Bcl-2/Bcl-xL and pro-
motes apoptosis [267]. However, unlike many other
known Bcl-2 family members, Bad ^ like Bid ^ has
no identi¢able C-terminal membrane anchor se-
quence and is probably not an integral membrane
protein. Its location in cells is dynamically controlled
by its association/dissociation with other Bcl-2 family
proteins [268]. The 1.1 kb bad mRNA is co-expressed
with Bcl-xL in many mouse tissues and two-hybrid
analysis and co-immunoprecipitation analyses indi-
cate that the Bad protein heterodimerizes with both
Bcl-2 and Bcl-xL but not with Bcl-xS , Bax, Mcl-1, A1
or itself. Experiments indicate that Bad counters the
death repressor activity of Bcl-xL , and probably ex-
erts its pro-apoptotic function by competing with
Bax for Bcl-xL binding.
Recently, regulation of Bad function has been
linked to phosphorylation [269]. In response to IL-
3 and other growth factors, Bad is phosphorylated at
three sites (Ser-112, Ser-136 and Ser-155); it then
binds to the cytosolic 14-3-3 protein and becomes
sequestered and functionally inactivated [270^277].
In contrast, regulation of another BH3-only do-
main protein, Bid [278], is regulated by proteolytic
cleavage rather than phosphorylation. Bid is cleaved
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F19
by caspase-8 and the truncated form then translo-
cates to mitochondria [279] where it activates Bax
[102,280] and Bak [103] by inducing oligomerization
of these pro-apoptotic Bcl-2 family members. Never-
theless, in HeLa cells treated with staurosporine to
undergo apoptosis, it is the full length Bid that trans-
locates to the mitochondria suggesting that caspase-
induced Bid cleavage is not an essential requirement
for its movement to mitochondria [280].
Recently, another BH3-only protein, Noxa, was
described to be regulated by P53 [281]. Like other
Bcl-2 family members it localizes to the mitochondria
after an apoptotic stimulus and is able to interact
with further Bcl-2 family members.
5.2.3. Bcl-2 family function
Although Bcl-2 has long been recognized as an
oncogene that acts as a suppressor of apoptosis,
the molecular basis for Bcl-2's anti-apoptotic action
has until recently been mysterious. Confocal and
electron microscopy studies show Bcl-2 to be local-
ized to the outer mitochondrial and nuclear mem-
branes and the ER. The mitochondrial localization
of Bcl-2 is perhaps especially intriguing in the light of
the emerging role of the mitochondrion in apoptosis.
At ¢rst this mitochondrial location fostered early
speculation that Bcl-2 might have some role in reg-
ulating oxidative phosphorylation [282,283], a notion
reinforced by the ability of Bcl-2 to suppress cell
death induced by oxidative damage, possibly by se-
questering free oxygen radicals. However, analysis of
cells grown under highly anoxic conditions [284] and
cells lacking mitochondrial DNA (and, hence, mito-
chondrial respiration) [285] indicated there was no
direct link between the function of anti-apoptotic
activity of Bcl-2 and oxidative phosphorylation.
Moreover, levels of cellular ATP or oxygen con-
sumption are not a¡ected by Bcl-2 [286,287].
Another possible mechanism by which Bcl-2 might
suppress apoptosis arose from observations that Bcl-
2 expression can a¡ect intracellular Ca2‡ homeosta-
sis [241]. Alterations in intracellular Ca2‡ concentra-
tions are known to in£uence PCD so it remains pos-
sible that Bcl-2 either directly modulates calcium
channels or acts to protect lipid membranes from
damage by peroxide radicals which is known to dis-
rupt Ca2‡ homeostasis.
Both Bcl-2 and Bcl-xL have been shown to interact
F20 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
directly with a variety of intracellular proteins.
Known interactors include pro-apoptotic Bcl-2 fam-
ily members (such as Bax [288], Bak and Bik), the
Raf-1 protein kinase, the protein phosphatase calci-
neurin, R-Ras and H-Ras, the P53 binding protein
P53-BP2, the prion protein Pr-1 and several other
proteins with unclear functions. The functional rele-
vance of many of these interactions is still a matter
of debate, e.g. the observed binding of Bcl-2 to the
C. elegans protein Ced-4 or its mammalian ortho-
logue Apaf-1. While some groups could show such
an interaction [93], others failed to do so [95,289].
The interaction between Bcl-2 and the pro-apoptotic
Bcl-2 family members is regarded as particularly im-
portant and will be discussed below.
Currently, three (non-exclusive) models are used to
explain Bcl-2 function. These models describe Bcl-2
proteins as ion channels, or as proteins that modu-
late activation of caspases, or as inhibitors of cyto-
chrome c export from mitochondria.
The interesting possibility that Bcl-2 family pro-
teins act as membrane channels emerges from eluci-
dation of the detailed three-dimensional structure of
Bcl-xL , which resembles the membrane insertion do-
main of bacterial toxins such as diphtheria toxin and
colicin [290]. Like Bcl-xL , these membrane insertion
domains contain two central helices consisting of
apolar residues that are able to span a membrane.
Because diphtheria toxin can form membrane pores
in a pH-dependent manner, it has been suggested
that Bcl-2 and its homologues act in a similar way
to generate pores in cytoplasmic and mitochondrial
membranes (possibly regulated by voltage- or pH-
dependent signals). Indeed, ion channel activity has
been detected with Bcl-2, Bcl-xL and with the pro-
apoptotic Bax protein in isolated lipid bilayers in
vitro, although at present there is no direct evidence
for in vivo channel formation [291,292]. Bax is able
to interact with VDAC [293] and/or the adenine nu-
cleotide translocator, ANT [294], to release cyto-
chrome c. After oligomerization it may also form
cytochrome c-releasing pores by itself [295]. This cy-
tochrome c release can be blocked by BH4 domain-
containing Bcl-2 family members such as Bcl-2 and
Bcl-xL [296].
A direct role for Bcl-2 proteins in regulating acti-
vation of apical (regulatory) caspases is indicated by
analogy with the basal apoptotic machinery of
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
C. elegans. In the nematode, the adapter protein
Ced-4 binds to Ced-3 [25,27,28] and induces ATP-
dependent autoactivation of the Ced-3 zymogen
[30,297]. Ced-9, the C. elegans homologue of Bcl-2,
physically interacts with Ced-4 and thereby blocks its
activation of Ced-3. Although nematode genetics has
identi¢ed only Ced-3, Ced-4 and Ced-9 as players in
this process, it is possible that there are other com-
ponents in such `apoptosome' complexes that cannot
be discriminated genetically by virtue of the fact that
they are essential proteins involved in some other
mandatory biological process ^ cytochrome c being
a candidate of this type. By analogy with the nem-
atode, Bcl-2/Bcl-xL may inhibit activation of the api-
cal caspase-9 by the Ced-4 orthologue Apaf-1. In
such a model, the role of pro-apoptotic Bcl-2 family
proteins is to displace Bcl-2/Bcl-xL from the Apaf-1/
cytochrome c/caspase-9 complex and so trigger cas-
pase-9 autoactivation [26].
In this context, the dynamics of Bax subcellular
localization are particularly interesting. In contrast
to Bcl-2/xL , Bax ^ at least in some cell types ^ ex-
hibits a di¡use localization throughout the cytosol.
However, upon induction of apoptosis, it rapidly re-
localizes to a punctate distribution that partially co-
localizes with mitochondria [298] where it presum-
ably associates with Bcl-2/Bcl-xL . This builds a
persuasive argument that relocalization of Bax is
part of a trigger for apoptosis, although the molec-
ular basis for its subcellular movement is still un-
known.
Biochemical analysis has shown that release of mi-
tochondrial cytochrome c is a central process in as-
sembly of the `apoptosome' ^ the multimeric com-
plex comprising caspase-9, Apaf-1, dATP and
cytochrome c that triggers autocatalytic cleavage
and activation of caspase-9 [37]. Cytochrome c nor-
mally resides in the intermitochondrial membrane
space, and the molecular mechanism of its transloca-
tion is not yet fully understood. One possibility is
that Bcl-2/xL , which resides on the outer surface of
the outer mitochondrial membrane, somehow acts to
forestall cytochrome c release [299^301].
It is possible that Bcl-2/xL proteins operate
through all three of these mechanisms. Perhaps, the
channel-forming activity of Bcl-2/xL proteins modu-
lates cytochrome c release whilst other regions of the
protein interact with Apaf-1. Interestingly, Bcl-2 it-
 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
self has been identi¢ed as a caspase target that is
cleaved during apoptosis to generate a carboxy-ter-
minal cleavage product that promotes apoptosis
[252]. This may be part of a positive feedback loop
within the cell death machinery.
For a long time the question has remained open
whether pro-apoptotic Bcl-2 family members are pro-
moting cell death as active molecules of the apoptotic
machinery or whether they rather neutralize anti-ap-
optotic Bcl-2 homologues which themselves represent
proteins actively participating in default survival
pathways. Recent data support the ¢rst scenario:
bax3=3 bak3=3 double knockout cells are resistant
towards radiation- and drug-induced apoptosis (in-
terestingly they remain sensitive towards CD95-medi-
ated cell killing) and overexpression of BH3-only
proteins that bind pro-survival Bcl-2 family members
fail to induce apoptosis in the absence of Bax and
Bak [104,263]. If the apoptotic Bcl-2 homologues are
indeed the `active' molecules while the role of anti-
apoptotic Bcl-2 family members is just to inhibit
them by binding, then Bcl-2, Bcl-xL etc. may not
be needed in the absence of Bax- and Bak-like mol-
ecules as seems to be the case in yeast.
Bcl-2 and other related family members not only
inhibit or promote apoptosis e¤ciently but also have
an in£uence on cell cycle progression. While over-
expression of Bcl-2, Bcl-xL , Epstein^Barr virus pro-
tein BHRF1, adenovirus E1B19kD or MCL-1 can
slow entry of cells into the cell cycle and promote
exit of cells from the cycle [302^305], overexpression
of the pro-apoptotic Bax protein can accelerate cell
cycle entry [306,307]. Some evidence suggests that the
anti-apoptotic function of Bcl-2 can be genetically
separated from its inhibitory e¡ect on cell cycle entry
[308]. The molecular basis for this cell cycle regula-
tion by Bcl-2 family members remains unclear but in
the case of MCL-1 the cell cycle regulatory function
is mediated through its binding of proliferating cell
nuclear antigen (PCNA), an interaction which seems
to inhibit PCNA's ability to promote progression
through S phase [305].
5.3. P53 and apoptosis
The p53 tumor suppressor gene is functionally in-
activated in 70% of human tumors [309]. P53 can
function as a transcription factor and the best char-
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F21
acterized activities of P53 are the induction of either
cell growth arrest or apoptosis [310]. Levels of P53
rapidly increase following DNA damage, mainly be-
cause the normally short-lived P53 protein becomes
stabilized, and this appears to be an important com-
ponent of the G1 arrest and apoptosis response that
follows DNA damage [311]. The induction of growth
arrest by P53 depends on its activity as a sequence-
speci¢c transcriptional activator and the P21
(WAF1/CIP-1) protein appears to be the major e¡ec-
tor of P53-mediated G1 cell cycle arrest after DNA
damage [311]. P21WAF1=CIPÿ1 binds to and inhibits
cyclin-dependent kinases, thereby blocking cell pro-
liferation.
How P53 mediates apoptosis is less clear and
seems to involve both transcriptional activation-de-
pendent and -independent pathways [312,313]. The
P21 cyclin-dependent kinase inhibitor is dispensable
for P53-dependent oncogene-induced apoptosis [312].
A di¡erent set of P53 target genes might regulate
growth arrest and apoptosis, respectively. Both func-
tions seem to be genetically dissectable: a human
tumor-derived P53 mutant was identi¢ed which has
speci¢cally lost its apoptotic function but not its cell
cycle function [314]. A model has been proposed in
which upstream e¡ectors (such as the atm gene prod-
uct which is defective in ataxia telangiectasia and
which is part of a pathway responding to DNA dam-
age as a result of ionizing radiation) selectively acti-
vate P53 to regulate speci¢c downstream pathways,
providing a mechanism for controlling distinct cell
cycle and apoptotic responses [315].
The decision whether an individual cell undergoes
growth arrest or apoptosis following P53 activation
appears to depend on a variety of factors, such as
environmental conditions and the cell type. It seems
safe to assume that, in the context of cellular DNA
damage following P53 cell cycle arrest, some `control
mechanism' evaluates whether the DNA can be re-
paired in a reasonable time or whether the damage is
so severe that the cell undergoes apoptosis in a P53-
dependent manner.
Analysis of P53-regulated gene expression patterns
using oligonucleotide arrays has demonstrated that
the nature of the P53 response in diverse mRNA
species depends on the levels of P53 protein in a
cell, the type of inducing agent or event, and the
cell type employed [316]. Of 6000 genes examined
F22 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
for P53 regulatory responses, 107 induced and 54
repressed genes fell into categories of apoptosis and
growth arrest, cytoskeletal functions, growth factors
and their inhibitors, extracellular matrix, and adhe-
sion genes.
Several mechanisms have been suggested by which
P53 protein might signal to the apoptotic machinery:
the pro-apoptotic Bcl-2 family member Bax appears
to be transcriptionally induced by P53 after DNA
damage in certain cell types [258]. However, Bax ap-
pears to contribute only in part to P53-mediated cell
death [241]: the pro-apoptotic P53 function seems
not to be e¡ected in bax-de¢cient mice and apoptosis
in transgenic Bax-expressing thymocytes is neither
increased nor accelerated in a p533=3 background.
In addition, genomic DMS footprinting has revealed
that the activity of the murine Bax promoter is regu-
lated by Sp1/3 and E-box binding proteins but not
by P53 [259].
In another scenario suggested for P53-mediated
cell death, both DNA damage and overexpression
of a wild-type p53 transgene cause transcriptional
induction of the TRAIL receptor DR5 through an
intronic sequence-speci¢c DNA binding site [317].
DNA damage fails to induce DR5 expression in tu-
mor cells with mutated p53. Interestingly, overex-
pression of DR5 (by transfection) appears to cause
apoptosis in a TRAIL ligand-independent manner,
implying that simply increasing the expression of
DR5 following P53 induction could result in activa-
tion of the apoptotic pathway [318].
UV- and X-ray radiation-induced apoptosis is
P53-dependent and seems to be mediated by activa-
tion of CD95 [319^321] providing another potential
link between P53 and a (cell surface) death receptor.
The CD95 receptor has already been suggested as a
target gene for transcriptional activation by P53
[322].
Other genes capable of inducing apoptosis have
been suggested as P53 targets: c-fos proto-oncogene
transcription is stimulated by P53 in cells undergoing
P53-mediated apoptosis [323] and MCG10 has been
identi¢ed as a novel P53 target gene encoding a KH
domain RNA binding protein capable of inducing
apoptosis and cell cycle arrest in the G2 ^M phase
of the cell cycle [324]. Very recently, Pidd, a new
death domain-containing protein, has been shown
to be induced by P53 and to promote apoptosis
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
[325]. This molecule might link death receptors
such as CD95 (which also contain a death domain
as a protein^protein interaction motif) to P53-medi-
ated cell death. Through direct cloning of P53 bind-
ing sequences from human genomic DNA, a novel
gene, designated p53AIP1 (P53-regulated apoptosis-
inducing protein 1), has been isolated, which is in-
ducible by wild-type P53 [326]. P53AIP1 is located
within mitochondria and its overexpression leads to
disruption of the mitochondrial membrane potential
and apoptotic cell death. Interestingly, phosphoryla-
tion of Ser-46 within P53 regulates the transcription-
al activation of this apoptosis-inducing gene.
While in general oncogene-induced apoptosis
seems to be P53-dependent [309] it is still a matter
of debate whether this is also true for Myc-induced
cell death. Although data from p53 knockout cells
support a P53 dependence of Myc killing [327] there
are other examples for P53-independent c-Myc-in-
duced apoptotic cell death [328].
P53 induces apoptosis not only through transacti-
vation-dependent but also through non-transcrip-
tional mechanisms. In human vascular smooth
muscle cells, P53 activation transiently increased sur-
face CD95 expression by transport from the Golgi
complex [329]. This cell surface redistribution of cy-
toplasmic CD95 transiently sensitizes cells to CD95-
induced apoptosis and occurs without new RNA
synthesis.
P53 can also contribute to apoptosis by direct sig-
naling at the mitochondria [330]: a small fraction of
stress-induced P53 protein after DNA or hypoxic
damage tra¤cs to mitochondria. This mitochondrial
localization precedes the release of cytochrome c and
procaspase-3 activation and is blocked by overex-
pression of anti-apoptotic Bcl-2 proteins. Redirecting
P53 from the nucleus and targeting it to mitochon-
dria by using mitochondrial import leader peptides is
su¤cient to induce apoptosis in P53-de¢cient cells,
even with a transcriptionally inactive p53 mutant.
As can be expected for an important protein such
as P53 and its pleiotropic signaling, regulation of P53
activity itself is rather complex and occurs at many
di¡erent levels [331,332]. A few examples are summa-
rized in the following paragraphs.
The oncoprotein Mdm2 binds to P53 and is a
physiological negative modulator of P53 activity.
Mdm2 can inhibit the function of P53 as a transcrip-
 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
tion factor by binding to the P53 N-terminus, there-
by preventing P53 from interacting with the tran-
scriptional machinery and inhibiting activation of
P53 responsive genes.
Mdm2 binding also targets P53 for ubiquitin-
mediated degradation. Upon DNA damage disrup-
tion of the Mdm2/P53 interaction is seen as a mech-
anism to increase P53's half-life in response to DNA
damage [331]. Mdm2 itself is regulated by the small
ubiquitin-like modi¢er protein SUMO-1: conjuga-
tion of Mdm2 with SUMO-1 excludes its self-ubiq-
uitination and increases Mdm1's ability to ubiquiti-
nate P53 [333]. Reduced Mdm2 sumoylation in
response to DNA damage contributes to P53 stabil-
ity.
The alternative reading frame (ARF) in the locus
encoding the Cdk inhibitor P16 can also bind to
Mdm2 and thus prevent Mdm2-mediated proteolytic
destruction of P53, although ARF-dependent mech-
anisms do not seem to be essential for the induction
of P53 levels in response to genotoxic stress
[334,335].
Other mechanisms exist to increase P53 levels: the
gene mutated in ataxia telangiectasia, ATM, is a ser-
ine/threonine kinase and P53 stabilization in re-
sponse to ionizing radiation is believed to occur in
part by ATM-dependent phosphorylation of P53 at
Ser-15, thereby disrupting the Mdm2/P53 complex
and increasing the half-life and transcriptional prop-
erties of P53 in a DNA damage-dependent fashion
[315,336]. The DNA-dependent protein kinase DNA-
PK is another candidate kinase for phosphorylating
P53 on Ser-15 [337]. As mentioned before, phosphor-
ylation of Ser-46 within P53 regulates transcriptional
activation of the pro-apoptotic P53 target gene
p53AIP1 [326]. P53-mediated cell growth arrest and
apoptosis is also modulated by acetylation and de-
acetylation [338].
Hypoxia (lack of oxygen in body tissue or tumor
mass) induces accumulation of wild-type P53
through hypoxia-inducible factor 1K (HIF-1K)-de-
pendent increase in p53 protein activity [339]: HIF-
1K binds to and stabilizes P53 but has no direct e¡ect
on P53 transcriptional activity.
Loss of the tumor suppressor Rb function may
contribute to P53-induced apoptosis [340]. In its hy-
pophosphorylated state, Rb binds to and inactivates
transcription factors of the E2F family. During the
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F23
cell cycle the Rb protein is phosphorylated by cyclin-
dependent kinases resulting in release of bound E2F
factors, which are necessary for the G1/S transition.
Loss of Rb results in uncontrolled proliferation and
P53-dependent apoptosis; this might explain why
many tumor types display mutations in both Rb
and p53 [340].
The P53 homologue P73 seems to play a role in
E2F-1-induced apoptosis [225,226]: activation of P73
provides a means for E2F-1 to induce death in the
absence of P53 and might constitute a P53-indepen-
dent, anti-tumorigenic safeguard mechanism.
6. Inhibition of apoptosis and tumorigenesis
Since the discovery of bcl-2 as an oncogene that
promotes cell survival it has been widely acknowl-
edged that anti-apoptotic genetic lesions are neces-
sary for tumors to arise. The net expansion of a
clone of transformed cells is not only achieved by
an increased proliferative index but also by a de-
creased apoptotic rate. The evidence is mounting,
principally from studies in mouse models and cul-
tured cells, as well as from descriptive analysis of
biopsied stages in human carcinogenesis, that ac-
quired resistance toward apoptosis is a hallmark of
most and perhaps all types of cancer [233,341,342].
Enhanced cell survival is needed at several steps
during tumorigenesis: deregulated oncogene expres-
sion not only leads to accelerated proliferation, but
concomitantly induces apoptosis which needs to be
suppressed for the transformed cell to survive and
multiply. Having reached a certain tumor size, su¤-
cient nutrition for every tumor cell becomes re-
stricted; starvation of (tumor) cells from cytokines
usually leads to apoptotic cell death. This selects
for further apoptosis resistance (and for angiogene-
sis). Finally, the metastasizing tumor cells, deprived
of cell^cell contact and of their normal (growth fac-
tor) environment, are prone to anoikis ^ further mu-
tations can suppress this cell death which occurs
when untransformed adherent cells detach from the
extracellular matrix.
An elegant transgenic mouse study provides in
vivo insight into at which stages during multistep
tumorigenesis apoptosis is downregulated [343].
Mice expressing the SV40 large T-antigen oncogene
F24 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37

in the L-cells of the pancreatic islets develop islet cell
carcinoma. Di¡erent stages during tumor develop-
ment can clearly be distinguished: an initial hyper-
proliferation involving 50^70% of the islets in the
pancreas is followed by an angiogenic phenotype
(10%) before ¢nally a few (1^2%) solid, encapsulated
tumors emerge. During this multistep process IGF-2
expression concomitant with the switch to hyperpla-
sia was shown to have a survival (rather than a mi-
togenic) function. Furthermore, transgenic overex-
pression of Bcl-xL led to an increase in tumor
incidence due to downregulation of apoptosis at the
conversion from the late preneoplastic state of angio-
genic islets to that of islet cell carcinoma.
Several other studies have shown that during tu-
mor progression apoptosis resistance is increasing,
for example in AKR lymphoma [344].
The following is a brief summary of data obtained
for the tumorigenic potential of some prominent pro-
teins known to be important for the regulation of
apoptosis (Fig. 3 summarizes current knowledge
about some apoptosis pathways involving proteins
which have been found in tumors to be deregulated).
6.1. Bcl-2 family
The Bcl-2 family has been implicated in tumori-
genesis for a long time. Bcl-2 was identi¢ed in human
follicular B-cell lymphoma where it becomes overex-
pressed as a consequence of a t(14;18) chromosomal
translocation. Bcl-2 synergizes e¡ectively with cell
proliferation-promoting oncogenes such as Myc
[233]: overexpression of Myc normally leads to in-
duction of apoptosis which is inhibited in vitro and
in vivo by Bcl-2 expression [345]. In addition to fol-
licular lymphoma, Bcl-2 levels are elevated in a
broad range of other human cancers including carci-
nomas of breast, prostate, ovary, colon, and lung,
indicating that this molecule might have a role in
raising the apoptotic threshold in a broad spectrum
of cancerous disorders [346].
In addition to its anti-apoptotic potential, Bcl-2
also inhibits entry into cell cycle, and the two Bcl-2
functions are genetically separable [308]. The anti-
proliferative behavior probably does not support tu-
mor growth and indeed loss of antimitotic e¡ects of
Bcl-2 with retention of anti-apoptotic activity during

Fig. 3. Signaling pathways involved in PCD. Overview of how di¡erent signaling pathways interconnect to regulate and determine the
apoptotic behavior of the cell. Molecules highlighted in red have been found mutated in tumor cells leading to increased resistance
against apoptotic stimuli.

BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart

 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
tumor progression has been observed in a mouse
model [347].
Overexpression of the anti-apoptotic Bcl-2 homo-
logue Bcl-xL enhances SV40 large T-antigen-medi-
ated transformation of pancreatic L-cells in transgen-
ic mice [343]. Bcl-xL is a transcriptional target gene
of the Stat-5 (and Stat-1 and Stat-3) transcription
factor [245,348]. Interestingly, Stat-5 is constitutively
phosphorylated and activated in Bcr/Abl-trans-
formed cells [349,350]. The bcr/abl oncogene is the
product of the t(9;22) translocation in chronic mye-
logenous leukemia. The resulting chimeric Bcr/Abl
protein displays enhanced tyrosine kinase activity
which leads to increased activation of Stat-5 and
elevated levels of its target gene Bcl-xL [349,351].
Expression of Bcr/Abl in hematopoietic cells induces
resistance to apoptosis in cell^cell and cell^matrix
interactions, thereby contributing to malignant pro-
gression by conferring a survival advantage.
Inactivation of pro-apoptotic Bcl-2 family mem-
bers may also contribute to tumorigenesis: a fraction
of human colon carcinomas with the mutator pheno-
type contain frameshift mutations in the bax gene
[352]. It has also been shown that P53-dependent
expression of Bax is induced in slow-growing apo-
ptotic transgenic mouse brain tumors; tumor growth
is accelerated and apoptosis drops by 50% in Bax-
de¢cient mice [262]. Another transgenic mouse study
showed that haploid loss of bax leads to accelerated
mammary tumor development in C3(1)/SV40 T-anti-
gen transgenic mice and that this increase in tumori-
genesis is due to a reduction in the protective apo-
ptotic response at the preneoplastic stage [353]. In
human colorectal cancer cells inactivation of Bax
completely abolished the apoptotic response to the
chemopreventive agent sulindac and other non-ste-
roidal anti-in£ammatory drugs in vitro [354]. These
results implicate Bax as a tumor suppressor.
On the other hand, Bax-de¢cient mice (without
any further tumorigenic transgene expression) do
not display an increased incidence of spontaneous
cancers and Bax de¢ciency does not further acceler-
ate oncogenesis in mice also de¢cient in P53 [355].
Surprisingly, transgenic overexpression of Bax in
T-cells in p53 knockout mice leads to accelerated
(rather than decreased) T-cell lymphomagenesis
although apoptosis was increased due to the pro-ap-
optotic function of Bax. In addition to promoting
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F25
apoptosis ^ and complementary to Bcl-2 ^ Bax in-
duces proliferation in T-cells, consequently Bax
transgenic mice have an increased percentage of cells
in cycle. Bax-induced proliferation seems to cooper-
ate in tumorigenesis with increased apoptosis resis-
tance due to P53 de¢ciency. Obviously, at least in
mice the dual roles of Bax can either accelerate or
inhibit tumorigenesis depending on the genetic con-
text.
For Bak, reduced protein expression (and elevated
Bcl-xL expression) has been demonstrated in primary
colorectal adenocarcinomas [356], a tumor type for
which the transformation of colorectal epithelium to
carcinomas had always been linked to inhibition of
apoptosis [357].
Because of functional redundancy between the
many Bcl-2 homologues the overall ratio between
the amount of pro- and anti-apoptotic Bcl-2 family
members might represent the important `rheostat' for
the cell's sensitivity towards apoptotic stimuli. Re-
lapse in childhood acute lymphoblastic leukemia
(ALL) for example is associated with a decrease in
the Bax/Bcl-2 ratio and with loss of spontaneous
caspase-3 processing in vivo [358]. The decreased
Bax/Bcl-2 ratio contributes to the observed distur-
bance of apoptotic pathways in ALL relapse.
6.2. TNF receptor family
Given the immunological importance and e¡ec-
tiveness of apoptosis induced by TNF receptor fam-
ily members one would expect their functional inac-
tivation to occur regularly during tumorigenesis
[359]. For example, expression of the soluble form
of CD95, which competes with the membrane-bound
receptor for natural ligand in human serum, corre-
lates with lymphoma (and autoimmune disease) and
loss of CD95 receptor accelerates lymphomagenesis
in EW L-Myc transgenic mice [360]. Of 150 cases of
human non-Hodgkin's lymphoma examined, 16 of
the tumors (11%) showed somatic CD95 mutations
indicating a link between CD95 receptor mutation,
cancer and autoimmunity [361]. Mutation analysis
for the entire coding regions of the CD95 gene
were performed in 43 cases of gastric cancer and
¢ve (11%) missense mutations were detected, all of
them in the death domain of the receptor [362]. In 81
de novo childhood T-lineage ALLs (T-ALL) exam-
F26 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
ined for the presence of CD95 mutations, one patient
with a heterozygous mutation in exon 3 of CD95
associated with decreased CD95-mediated apoptosis
was found [363]. In another study, CD95-de¢cient
transgenic mice overexpressing Bcl-2 develop acute
myeloblastic leukemia which again suggests that
CD95 may act as a tumor suppressor to control leu-
kemogenic transformation in myeloid progenitor
cells [364].
The anti-apoptotic protein c-FLIP inhibits death
receptor-induced apoptosis and was found to be
overexpressed in human melanomas. Two groups
have demonstrated that overexpression of c-FLIP
leads to immune escape of tumors in vivo presum-
ably by blocking CD95-mediated cytotoxic T-cell
killing [153,154,365]. Certain tumors may also escape
CD95 ligand-dependent immune-cytotoxic attack by
expressing a decoy receptor (DcR3) that blocks
CD95 ligand and its killing activity [155]. The
DcR3 gene was ampli¢ed and expressed in about
half of 35 primary lung and colon tumors studied.
Ampli¢cation and expression of DcR3 was also
found in Epstein^Barr virus- and HTLV-1-associated
lymphomas, probably allowing the virus to escape
from the immune system during lymphomagenesis
[366].
Recently, a new component of the CD95-DISC
has been identi¢ed, SADS (small accelerator for
death signaling), which enhances the interaction of
FADD and procaspase-8 and which is downregu-
lated in patients with colon carcinoma [367,368].
This downregulation most likely leads to an in-
creased resistance towards CD95-induced cell death
of the tumor cells (as observed with colon carcinoma
cells despite CD95 cell surface expression).
Nevertheless, although lpr and CD95knockout
mice develop a severe lymphoaccumulation, they do
not develop lymphoma and the relatively low percen-
tages of tumors with CD95 mutations identi¢ed in
the aforementioned studies do not support a role for
CD95 as a strong tumor suppressor a¡ected in a
large number of malignancies. Knockout mice for
other TNF receptor family members are not predis-
posed to tumorigenesis [233]. Perhaps the emerging
role for TNF receptor family members in prolifera-
tion prevents selection of their functional inactiva-
tion during tumor development at least in some types
of cancer [159].
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
CD95L expression is involved in another interest-
ing aspect of tumor biology: several tumors express
CD95L on their surface having downregulated their
own CD95 receptor expression. This might protect
the tumor cells against CD95L-induced apoptosis
while at the same time any activated T-cell bearing
CD95 on its surface and attacking the tumor would
be killed [369]. The same mechanism is used to es-
tablish immune privilege and could explain why can-
cer patients' immune systems fail to eliminate the
tumor. Nevertheless, the `tumor strikes back (by ex-
pressing CD95L)' model has been challenged recently
[370].
6.3. Caspases
Given the apparent central role of the caspase cas-
cade for the apoptotic pathway, it is striking that so
far functional inactivation of caspases has been iden-
ti¢ed in only a few tumors (although other mutations
either prevent caspase cleavage or directly inhibit ac-
tivated caspases, as discussed in this section). Cas-
pase-8 is frequently inactivated in neuroblastoma, a
childhood tumor of the peripheral nervous system
[371,372]. The gene is silenced through DNA meth-
ylation as well as through gene deletion, preferen-
tially in neuroblastoma with N-Myc ampli¢cations.
Caspase-8 null tumor cells are resistant to death re-
ceptor- and doxorubicin-mediated apoptosis. And
the resistance to TRAIL-induced apoptosis in prim-
itive neuroectodermal brain tumor cells correlates
with a loss of caspase-8 expression [373], again
showing that in principle caspase-8 can act as a tu-
mor suppressor. Decreased caspase-1 protein levels
have been reported as a potential step in the loss
of apoptotic control during prostate tumorigenesis
[374].
It is nevertheless worth noting that no cell type or
tumor cell line has been identi¢ed in which it is not
possible to eventually induce apoptosis via a certain
stimulus. This could be explained by a huge redun-
dancy in apoptosis-signaling pathways: for example,
mutation of any caspase would leave other caspases
capable of taking over the pro-apoptotic function
(furthermore, caspase-independent death pathways
seem to exist [85]). Alternatively it is possible that
proteins essential for induction and execution of ap-
optosis are simultaneously coupled to another vital
 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
cell function so that their inactivation leads to a le-
thal phenotype disallowing the selection for such mu-
tations [375].
6.4. IAPs
The IAP protein family plays an evolutionarily
conserved role in regulating PCD in diverse species
ranging from insects to humans [181]. The central
mechanisms of IAP apoptotic suppression appear
to be through direct caspase and procaspase inhibi-
tion (primarily caspases-9, -3 and -7) and modulation
of and by the transcription factor NF-UB. IAPs may
contribute to cancer by facilitating the insurgence of
mutations and by promoting resistance to therapy
[376]. The IAP family member survivin, while unde-
tectable in terminally di¡erentiated adult tissues, be-
comes prominently expressed in transformed cell
lines and in all the most common human cancers
of lung, colon, pancreas, prostate and breast [185].
Survivin is also found in approximately 50% of high-
grade non-Hodgkin's lymphomas [185,377] and high
survivin expression is signi¢cantly associated with
poor prognostic factors and promotes cell survival
in human neuroblastomas [378]. Another IAP family
member, cIAP-2, is known to undergo chromosomal
translocations and is activated in certain types of
lymphomas (MALTomas [379]). The caspase-inhibit-
ing and anti-apoptotic abilities of XIAP are blocked
by its binding partner, XIAP-associated factor 1
(XAF1) [380]. Cancer cell lines tested exhibited rela-
tively low xaf1 and high xiap mRNA levels com-
pared to normal tissue suggesting that a high level
of XIAP to XAF1 expression in cancer cells may
provide a survival advantage [381].
6.5. P53
P53 is mutated in most human tumors and the
resulting defects in (DNA damage-induced) growth
arrest and apoptosis have already been discussed.
Germ-line mutation of one p53 allele in human con-
fers a predisposition to develop various malignancies,
the Li^Fraumeni syndrome [233]. An impressive
demonstration of P53's role as a tumor suppressor
came from the p53 knockout mice which develop a
broad spectrum of tumors with a high incidence
[382]. Consequently, the MDM2 oncoprotein is over-
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
F27
expressed in 5^10% of human tumors. Its major
physiological role is to inhibit P53 and its anti-apo-
ptotic and growth-arresting activities [383].
Recent evidence indicates that in order for P53 to
exert its function as a growth arrest- and apoptosis-
inducing safety guard (i.e. as a tumor suppressor) it
needs to physically interact with another tumor sup-
pressor, ING-1 [384]. This interaction renders P53 an
even more e¡ective transcriptional activator (ING-1
can for example increase P53-dependent activation of
the p21/Waf-1 promoter). New data link histone ace-
tyltransferase activity to three yeast ING-1 homo-
logues, which suggests a role in chromatin remodel-
ing for ING-1 [385]. Therefore, loss of ING-1
function is another potential mechanism for the in-
activation of P53 in cancer cells.
Since Rb loss of function-induced apoptosis is
P53-dependent, both genes, p53 and Rb, are fre-
quently inactivated during cellular transformation.
Data on knockout mice have ¢rmly established that
germ-line mutations in p53 and Rb can cooperate in
tumorigenesis [386].
6.6. Other anti-apoptotic mutations implicated in
tumorigenesis
Apaf-1 expression is frequently lost due to meth-
ylation in metastatic melanoma tumors. Apaf-1 acts
with cytochrome c and caspase-9 to mediate P53-de-
pendent apoptosis [387,388]. Although p53 mutations
often occur in aggressive and chemoresistant cancers
they are rarely observed in melanoma. Apaf-1-nega-
tive melanomas are invariably chemoresistant despite
their functional P53 and they are unable to execute a
typical apoptotic program in response to P53 activa-
tion.
Gene silencing by methylation also plays a role in
the case of TMS1, a gene which is aberrantly meth-
ylated and silenced in 40% (11 of 27) of primary
breast tumors [389]. TMS1 encodes a 22 kDa protein
containing a terminal caspase recruitment domain
and its ectopic expression induces apoptosis in hu-
man breast cancer cells, placing TMS1 in the group
of pro-apoptotic tumor suppressor genes.
Overexpression of oncoproteins such as c-Myc,
c-Fos or c-Jun not only leads to proliferation but
simultaneously induces apoptosis. Consequently, ec-
topic overexpression of E2F-1 in glioma triggers ap-
F28 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
optosis and suppresses tumor growth in vitro and in
vivo [390]. The c-myc oncogene collaborates strongly
with bmi-1 in murine lymphomagenesis. While c-Myc
provides a strong proliferative stimulus, Bmi-1 inhib-
its c-Myc-induced apoptosis via P19ARF [391]. This
oncogenic collaboration is reminiscent of the Myc/
Bcl-2 collaboration in tumorigenesis where Bcl-2
also prevents Myc-mediated apoptosis [235].
In colon cancer, one of the earliest manifestations
is the formation of polyps, caused by somatic and
inherited mutations of the adenomatous polyposis
coli (APC) tumor suppressor gene in both humans
and mice. Overexpression of APC in human colorec-
tal cancer cells (which contain inactivated APC al-
leles) stops cell growth by inducing apoptosis, imply-
ing a role for suppression of apoptosis at the very
earliest stages of neoplasia [392].
The promyelocytic leukemia (PML) gene encodes
another tumor suppressor gene involved in the con-
trol of apoptosis. In most acute promyelocytic leuke-
mia patients, PML is fused to the retinoic acid re-
ceptor K (RARK) gene as a consequence of
chromosomal translocations. PMLRARK antago-
nizes PML function by heterodimerizing with PML
resulting in its delocalization from the nuclear body.
Inactivation of PML in hematopoietic cells leads to
increased resistance towards apoptotic stimuli and
lymphomagenesis [393].
Cells respond to a variety of stressful stimuli by
accumulating and/or activating a set of highly con-
served proteins known as heat shock proteins
(HSPs). These proteins normally function as molec-
ular chaperones by assisting the folding of newly syn-
thesized polypeptides, the assembly of multiprotein
complexes, and the transport of proteins across cel-
lular membranes [394]. Heat shock proteins such as
Hsp70, Hsp27 and Hsp90 can inhibit apoptosis
through direct physical interaction with key com-
ponents of the apoptotic machinery [395]. Hsp70
for example is highly expressed in human breast tu-
mors and tumor cell lines and antisense studies have
revealed that tumorigenic breast cancer cells depend
on the constitutive high expression of Hsp70 to sup-
press a transformation-associated death program
[394].
One molecule which could be responsible for sup-
pressing anoikis in metastasizing tumor cells is the
hyaluronan receptor CD44. CD44 has been impli-
BBACAN 87502 25-9-01 Cyaan Magenta Geel Zwart
cated in metastasis for a long time and it has been
demonstrated that disruption of cell surface CD44
function induces apoptosis in metastatic mammary
carcinoma cells in vivo [396].
6.7. Therapeutic approaches to induce apoptosis in
tumor cells
Identi¢cation of mutations in tumors that lead to
decreased apoptosis is not only of academic interest
but rather an important goal in the light of cancer
therapy. Clearly, mutations in cell death control do
a¡ect sensitivity of tumor cells to anti-cancer therapy
which in most cases functions by inducing apoptosis
[354,397]. Thus, for example, the e¡ectiveness of che-
motherapy might depend on the level of Bcl-2 ex-
pression in the tumor cells. The approach to decrease
Bcl-2 protein levels in tumor cells by applying Bcl-2
antisense oligonucleotides has already achieved
promising results in preclinical [398] and clinical
studies [399,400]. A similar antisense strategy tries
to induce apoptosis and to sensitize lung cancer cells
to chemotherapy by reducing survivin expression
[401].
Recently, a series of novel molecules have been
identi¢ed that inhibit the binding of the Bak BH3
peptide to Bcl-xL [402]. These chemical inhibitors
speci¢cally block the BH3 domain-mediated hetero-
dimerization between Bcl-2 family members and in-
duce apoptosis. Such small molecule inhibitors are
more stable than peptide inhibitors and are often
cell-permeable. Therefore they might represent prom-
ising new tools for cancer therapy.
TRAIL is a ligand for the apoptosis-inducing
death receptors DR4 and DR5. Given the remark-
able sensitivity of cancer cells compared with the re-
sistance in normal cells, recombinant TRAIL has
appealing therapeutic potential for a variety of hu-
man cancers [399]. Promising results have been ob-
tained in preclinical animal models involving human
tumor xenografts into SCID or nude mice. For glio-
mas, injected recombinant TRAIL caused complete
disease regression and entirely ablated tumor mass.
For colon carcinomas the combination of subthresh-
old doses of recombinant TRAIL with existing che-
motherapeutic agents resulted in a substantial posi-
tive interaction, completely eliminating the tumors in
some animals.
 M. Zo«rnig et al. / Biochimica et Biophysica Acta 1551 (2001) F1^F37
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