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Multiple cell death pathways as regulators of tumour initiation and
progression
Marja Ja
¨
a
¨
ttela
¨
*
,1
1
Apoptosis Laboratory, Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark
Acquired defects in signalling pathways leading to
programmed cell death (PCD) are among the major
hallmarks of cancer. Although focus has been on caspase-
dependent apoptotic death pathways, evidence is now
accumulating that nonapoptotic PCD also can form an
important barrier against tumour initiation and progres-
sion. Akin to the earlier landmark discoveries that lead to
the identification of the major cancer-related proteins like
p53, c-Myc and Bcl-2 as controllers of spontaneous and
therapy-induced apoptosis, numerous proteins with prop-
erties of tumour suppressors and oncoproteins have
recently been identified as key regulators of alternative
death programmes. The emerging data on the molecular
mechanisms regulating nonapoptotic PCD may have
potent therapeutic consequences.
Oncogene (2004) 23, 2746–2756. doi:10.1038/sj.onc.1207513
Keywords: cancer; caspase independent; cell death;
lysosome; transformation
Introduction
Cancer is a disease characterized by an imbalance
between cell division and cell death (Hanahan and
Weinberg, 2000). Apoptosis is the best-defined cell death
programme counteracting tumour growth. It is char-
acterized by the activation of a specific family of cysteine
proteases, the caspases, followed by a series of caspase-
mediated morphological changes such as the shrinkage
of the cell, the condensation of the chromatin and the
disintegration of the cell into small fragments that can
be engulfed by nearby cells without inciting inflamma-
tion (Kerr et al., 1972; Strasser et al., 2000; Ferri and
Kroemer, 2001; Kaufmann and Hengartner, 2001). With
respect to the ability of some cells to survive caspase
activation, it would be dangerous for the organism to
depend on a single protease family for the clearance of
unwanted and potentially harmful cells (Leist and
Ja
¨
a
¨
ttela
¨
, 2001). Indeed, accumulating data now show
that programmed cell death (PCD) can occur in the
complete absence of caspases, and evidence for death
pathways where noncaspase proteases and other death
effectors function as executioners is emerging (Ferri and
Kroemer, 2001; Leist and Ja
¨
a
¨
ttela
¨
, 2001; Lockshin and
Zakeri, 2002). Experiments employing cancer cells with
defective apoptosis machinery have revealed that most
caspase-activating apoptotic stimuli, including onco-
genes, p53, DNA-damaging drugs, proapoptotic Bcl-2
family members, cytotoxic lymphocytes and in some
cases even death receptors, do not require known
caspases for PCD to occur (Leist and Ja
¨
a
¨
ttela
¨
, 2001;
Mathiasen and Ja
¨
a
¨
ttela
¨
, 2002). Thus, in most death
pathways the role of caspases is rather at the level of
shaping the destruction of the cell that is already
committed to die than at the level of the decision to die.
In the following, I will first describe various types of PCD
and their control at molecular and organelle level and
then discuss the numerous links that connect transforma-
tion and tumour progression to the control of PCD.
Classification of PCD according to the nuclear
morphology
The foremost criterion for PCD (or active cell death)
that distinguishes it from accidental necrosis is the
participation of active cellular processes that can be
intercepted by interfering with intracellular signalling
(Leist and Ja
¨
a
¨
ttela
¨
, 2001). The unclear definition of
caspase-independent death pathways has been the major
obstacle to their better understanding. Since the precise
characterization of biochemical check points controlling
caspase-independent PCD is still awaiting, the nuclear
morphology of dying cells (Leist and Ja
¨
a
¨
ttela
¨
, 2001) has
been used as an alternative basis for the classification of
PCD (Figure 1). According to the nuclear morphology,
PCD can be divided into three subclasses: (i) classic
apoptosis characterized by chromatin condensed to
compact and almost geometric figures (stage 2 chroma-
tin condensation), (ii) apoptosis-like PCD with less
compact, lumpy chromatin masses (stage 1 chromatin
condensation) and (iii) necrosis-like PCD that occurs
either in the complete absence of chromatin condensa-
tion or at best with chromatin clustering to form loose
speckles (Leist and Ja
¨
a
¨
ttela
¨
, 2001). So far, nearly all
examples of classic apoptosis appear caspase dependent,
caspases playing an active role in the activation of DNA
fragmentation factor 45 (DFF45) that is responsible for
the compact chromatin condensation (Sakahira et al.,
1998). Theoretically, stage 2 chromatin condensation
*Correspondence: M Ja
¨
a
¨
ttela
¨
; E-mail: mhj@biobase.dk
Oncogene (2004) 23, 2746–2756
&
2004 Nature Publishing Group
All rights reserved 0950-9232/04 $25.00
www.nature.com/onc
could, however, occur caspase independently by
endonuclease G (EndoG), a mitochondrial DNase that
is released in response to various apoptotic stimuli (Li
et al., 2001). Most published forms of ‘caspase-
independent apoptosis’ fall into the category of apop-
tosis-like PCD even though some of the classic ‘caspase-
dependent apoptosis’ models also display this morphol-
ogy, for example due to the lack of DFF45 expression.
Apoptosis-inducing factor (AIF) released from the
mitochondria during various forms of PCD (Susin
et al., 1999), calcium-regulated cytosolic cysteine pro-
teases calpains, cathepsin B, a lysosomal cysteine
protease that translocates into the cytosol and nucleus
in response to many cellular stresses (Vancompernolle
et al., 1998), and
L-DNase II derived from the leucocyte
elastase inhibitor by post-translational modification
induced by low pH or digestion with elastase (Torriglia
et al., 1998), are among the effector molecules that can
trigger the stage 1 chromatin condensation. Autophagic
degeneration (also called type II PCD) (Chi et al., 1999)
and death receptor-induced necrosis (Vercammen et al.,
1998a) lack chromatin condensation and can thus be
classified as necrosis-like PCD. Owing to the enormous
overlap and shared signalling pathways between the
different death programmes, cells dying by apoptosis-
and necrosis-like PCD can display any degree and
combination of other apoptotic features including
phosphatidylserine exposure, cytoplasmic shrinkage,
zeiosis, formation of apoptotic bodies, and even the
activation of caspases (Leist and Ja
¨
a
¨
ttela
¨
, 2001). It
should also be noted that a single stimulus often triggers
several distinct death programmes concurrently. Nor-
mally, only the fastest and most effective death pathway
is evident, but one cell may also display characteristics
of several death programmes simultaneously (Bursch
et al., 1996).
Classification of PCD according to the involvement of
cellular compartments
The classification of PCD based on the nuclear
morphology does not take into the account the death
signalling pathways involved. Therefore, attempts to
sort PCD according to the cellular compartments
involved in the process (mitochondria, lysosomes,
plasma membrane receptors, nuclei, cytoskeleton and
endoplasmic reticulum) may provide a more informative
basis for the taxonomy (Ferri and Kroemer, 2001). It
does, however, not offer a simple solution, because most
death pathways depend on input from several parts of
the cell, the major one being the mitochondrion that is
essential for the vast majority of death pathways
(Figure 1). As mitochondrial membrane permeabiliza-
tion (MMP) is the event that defines the point of no
return in most PCD models, its control and conse-
quences are described first followed by the depiction of
the involvement of other cellular compartments to
MMP-dependent as well as MMP-independent PCD.
Control of MMP
The pathways upstream of MMP are numerous and
with only few exceptions tightly controlled by the
members of the Bcl-2 family (Coultas and Strasser,
2003) (Figure 1). Bax and Bak, the ‘multidomain’
proapoptotic Bcl-2 family members, are crucial pore-
forming molecules that trigger MMP and the release
of death-inducing molecules from the mitochondrial
intermembrane space. Bax/Bak induce initially outer
Figure 1 The control and consequences of MMP. Pore-forming
proteins Bax and/or Bak can trigger MMP following activation by
BH3-only proteins or cleavage. Caspase-8 and cysteine cathepsin
can cleave and activate a BH3-only protein, Bid, disruption of the
cytoskeleton leads to the release of BH3-only proteins Bim and
Bmf, activation of the JNK pathway increases the expression Bmf
and Hrk, DNA damage induces a p53-mediated transcription of
genes encoding Bax, BH3-domain only proteins (Noxa and
PUMA) as well as proteins involved in ROS generation, ER stress
results in the release of Ca
2 þ
, which may cause direct mitochondrial
damage or activate Bax via calpain-mediated cleavage and
hypoxia-activated transcription factor HIF-1a induces the expres-
sion of BNIP3, whose stability is increased by acidosis, and
activation of CD47 releases BNIP3 from the receptor allowing it to
translocate to the mitochondrial membrane. Furthermore, various
death stimuli trigger the production of lipid second messengers that
are involved in MMP and mitochondrial damage. Mitochondrial
damage leads to the release of numerous mitochondrial proteins
that trigger the execution of PCD. Cytochrome c, Smac/Diablo and
Omi/Htra2 triggers caspase activation and classic apoptosis. AIF
triggers a caspase-independent death pathway culminating in DNA
fragmentation and stage 1 chromatin condensation characteristic
of apoptosis-like PCD. Endonuclease G cleaves DNA and induces
stage 2 chromatin condensation. The serine protease activity of
Omi/Htra2 mediates caspase-independent cellular rounding and
shrinkage without changes in the nuclear morphology. Ca
2 þ
and
ROS can lead into severe mitochondrial dysfunction and necrosis-
like PCD with or without autophagy. The schematic drawings in
the bottom of the figure provide a general guideline to differentiate
between the different PCD forms according to the nuclear
morphology. Caspase-dependent chromatin compaction and frag-
mentation to crescent- or spherical-shaped masses at the nuclear
periphery is shown in the middle. Caspase-independent chromatin
margination triggered directly by microinjection of AIF or in a
number of other models of apoptosis-like death is shown at right.
No chromatin condensation, but sometimes passive clumping and
dissolution of nucleoli is characteristic of necrotic PCD
Cell death pathways as regulators of tumour initiation and progression
MJa¨a¨ttela¨
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membrane permeability and leave the inner membrane,
the protein import function and the ultrastructure of
mitochondria intact (Von Ahsen et al., 2000). Bax/Bak
can be activated transcriptionally or by conformational
change induced by cleavage or binding to BH3-only
proteins (a subgroup of proapoptotic Bcl-2 proteins that
share homology to Bcl-2 only in the Bcl-2 homology
domain 3). Antiapoptotic Bcl-2 proteins (e.g. Bcl-2 and
Bcl-X
L
) oppose MMP presumably by heterodimeriza-
tion with Bax-like proteins. In contrast, proapoptotic
‘BH3-only’ proteins (Bad, Bid, Bim, Bmf, Noxa,
PUMA, BNIP3, etc.) either oppose the inhibitory effect
of Bcl-2-like proteins via BH3-mediated binding or
activate Bax-like proteins by direct binding. BNIP3, a
death-promoting ‘BH3-only’ protein differs from the
other proteins of this class. It can integrate into the
mitochondrial outer membrane in a BH3-domain- and
presumably also Bax/Bak-independent manner to trig-
ger the loss of mitochondrial transmembrane potential,
reactive oxygen species (ROS) generation and PCD with
characteristics of autophagic degeneration (Vande Velde
et al., 2000). Similarly, overexpression of Hspin1,
another Bcl-2 binding transmembrane protein with no
BH3 domain, causes upregulation of a lysosomal
protease cathepsin D and autophagy without release of
proapoptotic proteins from the mitochondrial inter-
membrane space (Yanagisawa et al., 2003). Further-
more, increase in cytosolic calcium and arachidonic acid
may induce mitochondrial damage in a Bax/Bak-
independent manner (Scorrano et al., 2001; Scorrano
et al., 2003).
Consequences of MMP
MMP limited to the mitochondrial outer membrane is
sufficient for the release of death-promoting molecules
from the mitochondrial intermembrane space, whereas
permeabilization of both membranes normally results in
necrosis-like PCD characterized by the loss of mito-
chondrial transmembrane potential and major meta-
bolic disturbances due to the reduced ATP generation
and increased production of ROS (Figure 1). The
classical apoptosis pathway is initiated by the release
of cytochrome c (Li et al., 1997). It prompts the ATP-
dependent assembly of the apoptosome complex con-
sisting of cytochrome c, apoptotic protease-activating
factor 1 and caspase-9, which forms the template for
efficient caspase processing and activation. As a further
safe-guard mechanism, caspase-inhibitory factors (pro-
teins of inhibitor of apoptosis protein family) have to be
removed by additional proteins (Smac/Diablo or Omi/
htra2) released from mitochondria before the execution
caspases can become fully active and bring forth the
typically apoptotic morphology best characterized by
the compact condensation of the chromatin (Du et al.,
2000; Suzuki et al., 2001).
Another distinct pathway following MMP is mediated
by AIF (Susin et al., 1999). Once released from the
mitochondria, AIF translocates into the nucleus where it
induces caspase-independent type 1 chromatin conden-
sation by an as yet undefined mechanism. EndoG and a
serine protease Omi/Htra2 may also contribute to the
caspase-independent death signalling downstream of
MMP. Extramitochondrial expression of Omi triggers
necrosis-like PCD that is dependent on Omi’s serine
protease activity (Suzuki et al., 2001), and EndoG can
cause caspase-independent DNA fragmentation in iso-
lated nuclei (Li et al., 2001). Caenorhabditis elegans AIF
ortholog (wah-1) associates and cooperates with the C.
elegans EndoG ortholog (CPS-6) to promote DNA
degradation and apoptosis (Wang et al., 2002). Whether
EndoG and AIF also define a single MMP-initiated
DNA degradation pathway in mammalian cells remains
to be studied. Whereas there is emerging evidence
suggesting that AIF may serve as a safe-guard death
executioner in some cancer cells with faulty caspase
activation in vitro as well as in c-Myc-induced mammary
tumours in vivo (Joseph et al., 2002; Alonso et al., 2003;
Liao and Dickson, 2003), the roles of EndoG and Omi/
Htra2 in cancer cell death await to be elucidated.
Instead of the relatively rapid death pathways
described above, mitochondrial damage may also lead
to a slow sequestration of the damaged mitochondria in
autophagic vacuoles (Tolkovsky et al., 2002). If the
damage is limited, autophagy may serve as a rescue
mechanism inhibiting the further release of the death-
promoting mitochondrial proteins to the cytosol. How-
ever, in the case of more severe or continuous damage,
autophagy leads to metabolic death after a lag phase of
several days.
In the majority of cell death models, the master
controllers of PCD operate at the mitochondrial level,
whereas the decision concerning the nature of the death
is taken after this step. There are, however, certain cases
where mitochondria may not hold a regulatory role, but
instead organelles like lysosomes and endoplasmic
reticulum (ER) might be in charge of the execution of
the cell (Chi et al., 1999; Elliott et al., 2000; Nylandsted
et al., 2000; Sperandio et al., 2000; Inbal et al., 2002;
Nylandsted et al., 2002).
Receptor-mediated PCD comes in many shapes
Plasma membrane plays an important role in the life-
death decisions of the cell via its numerous receptors
that promote either survival or PCD. Death receptors of
tumour necrosis factor receptor (TNFR) superfamily,
for example TNFR1 and Fas (also known as CD95) and
receptors for TNF-related apoptosis-inducing ligand
(TRAIL), trigger almost the whole spectrum of different
death pathways either dependent or independent of
MMP and caspases and provide thus an exceptional
example of the complexity of PCD signalling (Ashke-
nazi, 2002; Ja
¨
a
¨
ttela
¨
and Tschopp, 2003). The basic
signalling unit of DRs consists of three receptors, which
are brought together and actived upon binding to the
trimeric ligand. The receptors then assemble a death-
inducing signalling complex. Through a death domain-
mediated binding of the adaptor proteins TRADD
(TNF-R1) and FADD (all death receptors), death
receptors recruit and activate the apoptosis-initiating
proteases caspase-8 and/or caspase-10 that induce
Cell death pathways as regulators of tumour initiation and progression
MJa¨a¨ttela¨
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apoptosis either by direct activation of effector caspases
or via a Bax/Bak-dependent MMP triggered by caspase-
8-mediated cleavage of Bid (Luo et al., 1998; Scaffidi
et al., 1998). Another type of signalling complex induced
by death receptors involves the kinase receptor-asso-
ciated protein 1 (RIP1) and the caspase-8 inhibitor
FLIP, which, via their interaction with TNFR-asso-
ciated factors (TRAFs) links death receptors to the
signalling cascades activating the NF-kB transcription
factor and mitogen-activated protein kinases (Thome
and Tschopp, 2001).
Embryonic fibroblasts or thymocytes from mice
deficient in FADD and caspase-8 are resistant to FasL
and TNF (Varfolomeev et al., 1998; Yeh et al., 1998;
Zhang et al., 1998), revealing the central role of caspase
activation and thus apoptosis in death receptor signal-
ling. A number of recent reports show however that
FasL, TNF and TRAIL cause cell death even in the
absence of caspase activation in other cell types. The
nonapoptotic caspase-independent PCD may even be
predominant, for instance in the L929 fibrosarcoma cell
line (Vercammen et al., 1998a, b). These cells respond to
TNF with necrosis-like PCD without the involvement of
caspases, despite the fact that Fas-stimulation leads to
caspase-dependent apoptosis. When caspases are inhib-
ited by various means, the cells still die upon Fas
stimulation, albeit with reduced efficacy, delayed ki-
netics and necrosis-like morphology. Thus, within the
same cell line both death pathways are available to
death receptors but are differentially engaged depending
on the nature of the incoming signal. Since the presence
of caspase inhibitors in L929 cells results not only in the
appearance of a Fas-triggered necrosis, but also in a
1000-fold sensitization towards TNF-induced necrosis,
caspases may be implicated in an antinecrotic pathway.
The DR-induced caspase-independent pathway is not
restricted to the L929 cells. In the U937 and Jurkat
leukaemia, HT29 colon carcinoma, NIH3T3 fibroblast
and WEHI-S fibrosarcoma cells, caspase-independent
PCD with either necrosis- or apoptosis-like morphology
can be engaged by TNF, FasL and/or TRAIL (Khwaja
and Tatton, 1999; Holler et al., 2000; Luschen et al.,
2000; Foghsgaard et al., 2001; Wilson and Browning,
2002). Our understanding of the signalling pathway
leading to DR-induced nonapoptotic PCD and the
mechanisms governing the decision points between the
caspase-dependent and -independent cell death is as yet
limited. FasL-induced necrotic PCD is absent in T cells
that are deficient in FADD, indicating that the presence
of FADD is crucial for both necrotic and apoptotic
PCD induced by FasL (Holler et al., 2000). Another
protein involved in the necrotic signalling cascade is
RIP1. Jurkat cells deficient in RIP1 are completely
resistant to cell death even at high concentrations of
FasL, provided that caspases are inhibited (Holler et al.,
2000). Unlike RIP1’s function in the recruitment of the
activation of NF-kB (Kelliher et al., 1998), the kinase
activity of RIP1 is indispensable for its death-promoting
activity (Holler et al., 2000). This suggests that RIP1
phosphorylates and thereby regulates a key player of the
nonapoptotic cell death programme. Fas can also trigger
caspase-independent apoptosis-like PCD. In immorta-
lized epithelial cells, activated Fas recruits Daxx from
the nucleus to the receptor complex and triggers its
binding with apoptosis signal-regulating kinase 1
(Ask1). In addition to a caspase-dependent proapopto-
tic function that depends on its kinase activity, Ask1
possesses a caspase-independent killing function that is
independent of its kinase activity and is activated by
interaction with Daxx (Charette et al., 2001). The
relationship between RIP1-mediated and Daxx/Ask1-
mediated death pathways remains to be studied.
The caspase-independent PCD triggered by TNF is
mechanistically similar but not identical to that trig-
gered by Fas. While the kinase activity of RIP1 is also
obligatory, FADD appears to be dispensable (Holler
et al., 2000). Furthermore, pharmaceutical inhibition
and antisense cDNA-mediated depletion of the lysoso-
mal cysteine protease cathepsin B confers almost
complete protection against TNF (and TRAIL)-induced
apoptosis-like caspase-independent PCD in WEHI-S
cells (Foghsgaard et al., 2001). Consistent with the
prominent role of cathepsin B in this pathway,
immortalized murine embryonic fibroblasts (iMEFs)
deficient for cathepsin B are highly resistant to TNF
(Foghsgaard et al., 2002). Interestingly, cathepsin B-
deficient primary MEFs display similar sensitivity to
TNF as the wild-type MEFs (Foghsgaard et al., 2001),
but upon cellular immortalization MEFs become over
1000-fold sensitized to TNF-induced apoptosis-like
PCD that is cathepsin B-dependent and caspase-
independent. This sensitization is further increased upon
Ras- and Src-mediated transformation of iMEFs (N
Fehrenbacher and MJ, unpublished). Whether cathepsin
B is on the same pathway as RIP1 is still an open
question. This is, however, supported by data showing
that the treatment of Jurkat cells with TLCK, a potent
inhibitor of serine and cysteine proteases including
cathepsin B, inhibits RIP1-dependent necrosis-like PCD
induced by TNF and FasL (Holler et al., 2000).
There is ample evidence that excess ROS formation is
involved in the nonapoptotic PCD induced by death
receptors. Inhibition of caspases (which sensitizes to
necrosis) results in increased ROS formation and the
addition of butylated hydroxyanisole (BHA), an oxygen
radical scavenger, hinders both TNF- and FasL-induced
necrosis-like PCD in L929 cells and TNF-induced
apoptosis-like PCD in WEHI-S cells and iMEFs
(Vercammen et al., 1998b; Foghsgaard et al., 2002).
TNF-induced ROS has been proposed to be a result of
enhanced electron flow through the mitochondrial
electron transport chain (Schulze-Osthoff et al., 1992).
Other possible intracellular sources of ROS include
lysosomes and phospholipase A2 (PLA2). Interestingly,
the TNF-induced activation of PLA2 is dependent on
cathepsin B (Foghsgaard et al., 2002).
In addition to the death pathways described above,
TNF-R1 triggers caspase-independent autophagy and
membrane blebbing mediated by death-associated pro-
tein kinase (DAPK) and DAPK-related protein kinase 1
(DRP-1) in cells displaying caspase-mediated and
DAPK/DRP1-independent nuclear fragmentation
Cell death pathways as regulators of tumour initiation and progression
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(Inbal et al., 2002). Interestingly, inhibition of caspases
but not DAPK/DRP1 could rescue cells from death. In
fact DAPK/DRP1 inhibition increased the amount of
cells with condensed and fragmented nuclei, suggesting
that autophagy may function as a rescue mechanism
against caspase-mediated apoptosis in this model system.
Other cell surface receptors that trigger alternative
death pathways in a cancer-specific manner include
CD20, CD47, CD99 and insulin-like growth factor 1
receptor (IGF1R). Rituximab is an anti-CD20 antibody
successfully used for the treatment of lymphoma. In
vitro, it induces a mitochondria- and caspase-indepen-
dent exposure of phosphatidylserine and caspase-inde-
pendent loss of mitochondrial membrane potential,
suggesting that its efficacy may be due to the ability to
kill lymphoma cells in spite of defective caspase
activation or overexpression of Bcl-2-like proteins
(Chan et al., 2003). Activation of CD47, a receptor for
thrombospondin, initiates necrosis-like PCD in B-cell
chronic lymphoma cells (Mateo et al., 1999). Also,
resting B and T cells are affected but to a lesser degree
suggesting that therapies targeting CD47 may prove
useful in the clinic (Mateo et al., 2002; Lamy et al.,
2003). CD47-initiated death is characterized by early
mitochondrial dysfunction, suppression of mitochon-
drial membrane potential and generation of ROS
without signs of cytochrome c release or caspase
activation. BNIP3, the Bcl-2 family protein mediating
autophagy (Vande Velde et al., 2000), may be the
effector molecule in this pathway (Lamy et al., 2003). It
binds to the unoccupied receptor, but translocates to the
mitochondrial membrane upon ligand or antibody
binding. Furthermore, downregulation of BNIP3 with
antisense oligonucletides partially attenuates CD47-
induced necrotic death. Antibodies to yet another
lymphoid receptor, CD99 triggers a rapid and profound
apoptosis-like PCD in transformed but not normal T
cells by an as yet unidentified pathway (Pettersen et al.,
2001).
Ligand-activated IGF1R mediates one of the most
potent survival signals for cancer cells (Evan and
Vousden, 2001). Overexpression of an unoccupied
IGF1R or its cytosolic tail triggers, however, necrosis-
like death that is independent of MMP, Apaf-1 and
caspase activity (Sperandio et al., 2000). Surprisingly,
this pathway is dependent on procaspase-9 protein
suggesting that procaspase-9 may posses another enzy-
matic activity or function as an adaptor protein linking
unidentified death effectors to IGF1R in order to trigger
necrosis-like PCD in cells deprived of IGF. Thus,
IGF1R antagonists may turn out to be more successful
in cancer therapy than anticipated. Furthermore, it will
be interesting to study whether other so-called depen-
dence receptors activate a similar pathway. In the case
of ‘deleted in colorectal cancer’ (DCC), an Apaf-1- and
MMP-independent death pathway has already been
described, but contrary to IGF1R, DCC-induced
apoptosis is dependent on caspase activity (Forcet
et al., 2001). Activation of this pathway may, however,
prove useful in treatment of cancers with defects in the
mitochondrial pathway.
Lysosomes, the underestimated suicide bags
Lysosomes have until recently been considered as
‘suicide bags’ that through the release of unspecific
enzymes cause autolysis and damage neighbouring cells
during uncontrolled tissue damage. However, accumu-
lating data now show that lysosomes also function as
death signal integrators in many controlled death
paradigms (Brunk et al., 2001; Ferri and Kroemer,
2001; Turk et al., 2002; Ja
¨
a
¨
ttela
¨
and Tschopp, 2003)
(Figure 2). Lysosomal proteases, cathepsins, translocate
from the lysosomal lumen to the cytosol in response to a
wide variety of death stimuli such as TNF and TRAIL
(Foghsgaard et al., 2001), Fas (Brunk and Svensson,
1999), p53 activation (Yuan et al., 2002), retinoids
(Zang et al., 2001), growth factor starvation and
oxidative stress (Brunk and Svensson, 1999), B-cell
receptor activation (van Eijk and de Groot, 1999), T-cell
activation and staurosporine (Bidere et al., 2003). Once
released to the cytosol, cathepsins, especially cysteine
cathepsins B and L and aspartyl cathepsin D, may
trigger MMP followed by caspase- or AIF-mediated
apoptosis (Roberg et al., 2002; Bidere et al., 2003; Boya
et al., 2003; Cirman et al., 2003). Akin to caspase-8,
lysosomal cysteine cathepsins can cleave and activate
Bid (Cirman et al., 2003). Thus, Bid may function as one
of the links that connects lysosomal leakage to MMP.
Lysosomal leakage may also trigger mitochondrial
dysfunction in a Bid- and Bcl-2-independent manner
Figure 2 Lysosomal control of PCD. Lysosomal leakage is
induced by the indicated stimuli by an undefined mechanism.
Effector molecules released from the lysosome include cathepsins,
ROS and H
þ
. Cathepsins released from the lysosomes function in
the apoptosis pathway by causing mitochondrial membrane
permeabilization via cleavage of Bid or via activation of PLA2
and the following increase in arachidonic acid (AA) level.
Cathepsins may also directly cause caspase-independent chromatin
condensation. Acidification of the cytosol can stabilize BNIP3 that
triggers autophagy or activate
L-DNase II that can cause partial
chromatinolysis in a caspase-independent manner. ROS may be
involved in mitochondrial damage and necrosis-like PCD
Cell death pathways as regulators of tumour initiation and progression
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(Boya et al., 2003). Second messengers involved in such
pathways may include ROS and arachidonic acid (Boya
et al., 2003; Foghsgaard et al., 2002). Cathepsins can
also mediate MMP- and caspase-independent PCD with
apoptosis-like morphology (Vancompernolle et al.,
1998; Foghsgaard et al., 2001). As the latter pathway
can circumvent most of the known resistance mechan-
isms occurring in tumour cells, lysosomal membrane
permeabilization appears as a promising target for
cancer therapy.
Lysosomal leakage leads inevitably to intracellular
acidification, whose role in lysosomal PCD has been
only poorly studied. The acidification of the cytosol has,
however, many consequences that may contribute to the
lysosomal PCD. Firstly, acidification has been shown to
trigger cJun N-terminal kinase (JNK)-mediated caspase-
and Bcl-2-independent necrosis-like PCD in bladder
cancer cells (Zanke et al., 1998). Secondly, acidification
can induce a post-translational modification of an
antiapoptotic serine protease inhibitor, leucocyte elas-
tase inhibitor, that converts it into
L-DNase II that in
turn can mediate nuclear changes typical of apoptosis-
like PCD (Altairac et al., 2003). And finally, autophagy-
inducing Bcl-2 family member BNIP3 can be stabilized
by intracellular acidification (Kubasiak et al., 2002).
Whether these mechanisms are involved in lysosomal
death pathways needs to be experimentally tested, but
they may explain some of the varying morphologies seen
following lysosomal leakage.
Lysosomes are also crucial in the autophagic degen-
eration (Lockshin and Zakeri, 2002; Reggiori and
Klionsky, 2002; Ogier-Denis and Codogno, 2003). Even
though autophagy can be initiated by mitochondrial
damage, it does also occur in the absence of MMP, and
most described forms of autophagic death are insensitive
to Bcl-2. During autophagy (or macroautophagy), a
portion of cytosol or cytosolic organelles are sequestered
by a structure known as autophagosome that is isolated
from the remaining cell contents by a double-membrane.
Subsequently, the autophagosome fuses with lysosomes
and the inner membrane as well as the sequestered
materials are digested by lysosomal enzymes. Morpho-
logically, autophagy is characterized by the appearance
of abundant vacuoles in the cytosol and it has been
described as a prominent form of PCD during develop-
ment (Lockshin and Zakeri, 2002). Of interest to cancer
research, autophagy-inducing stimuli include starvation,
hypoxia, radiation, antioestrogens as well as cytokines
such as TNF and interferon g (Ogier-Denis and
Codogno, 2003). Moreover, emerging data suggest that
autophagy may play a central role in the control of
tumorigenesis (see below).
Nucleus as a death sensor
The best-defined nuclear death pathway leading to
MMP and apoptosis is that triggered by DNA
damage-induced activation of p53 (Vousden and Lu,
2002). Bax and ‘BH3-only’ proteins Noxa and PUMA
are direct target genes of p53 and gene knockout studies
have revealed that they are required for efficient DNA
damage-induced MMP and apoptosis (Villunger et al.,
2003). Additionally, ROS may mediate p53-induced
MMP via the upregulation of genes involved in ROS
generation. Recent data also suggest that p53 may serve
as a link to the stress-induced direct activation of
caspase-2. p53-induced death domain protein (PIDD),
RAIDD (RIP-associated ICE-like death domain pro-
tein) and caspase-2 form an apoptosome-like complex
capable of activating caspase-2 (J Tschopp, personal
communication). The activated caspase-2 can then act
as an initiator caspase and trigger MMP to amplify the
caspase activation (Lassus et al., 2002). Whether the
stress-induced caspase-2 can mimic DR-activated cas-
pase-8 and engage a lethal caspase activation cascade
independent of MMP is as yet unclear.
Studies employing various pharmacological protease
inhibitors suggest that also noncaspase proteases with
activities resembling cysteine cathepsins (inhibited by z-
FA-fmk) and possibly other cysteine or serine proteases
(inhibited by TLCK) participate in p53-mediated PCD
(Lotem and Sachs, 1996). This may be explained by the
recent data showing that p53 provokes an early
lysosomal leakage that precedes MMP (Yuan et al.,
2002). Inhibitors of lysosomal cysteine proteases block
MMP in this model, suggesting that lysosomal leakage is
an early event that is necessary for the p53-induced
PCD. Additionally, DNA damage induced by g irradia-
tion, etoposide or adriamycin upregulates lysosomal
aspartyl protease cathepsin D in a p53-dependent
manner, and pepstatin A, a cathepsin D inhibitor,
partially suppresses p53-dependent death of lymphoid
cells (Wu et al., 1998). p53-induced lysosomal leakage is
not attenuated by an antioxidant implicating that ROS
are not involved in this process, whereas the ability of
caspase-2 and proapoptotic Bcl-2 family members to
destabilize lysosomes remains to be elucidated.
In spite of their importance in respect to the treatment
of human cancer, p53-independent apoptotic responses
to DNA damage are poorly studied. Possible pathways
involve activation of other tumour suppressor proteins;
for example, interferon regulatory factor 1 (Tamura
et al., 1995), breast cancer susceptibility gene BRCA1
(Harkin et al., 1999), p73 (Gong et al., 1999) and
promyelocytic leukaemia protein (PML) (Yang et al.,
2002). In addition to caspase-mediated apoptosis, PML
can trigger a caspase-independent death programme
displaying cytoplasmic apoptotic features, which are
even enhanced in the presence of pan-caspase inhibitors
(Quignon et al., 1998; Wang et al., 1998). Recent results
show also that DNA-damaging agents can trigger
lysosomal leakage in cancer cells lacking functional
p53 (J Nylandsted and MJ, unpublished).
ER as a sensor of stress
ER is an important sensor of cellular stress and it may
initiate PCD by at least two distinct mechanisms, the
unfolded protein response and release of Ca
2 þ
(Ferri
and Kroemer, 2001). Both events lead eventually to
MMP and may thus activate the classical apoptosis
pathway as well as the other death pathways triggered
Cell death pathways as regulators of tumour initiation and progression
MJa¨a¨ttela¨
2751
Oncogene
by MMP. Stimuli that induce an increase in the
intracellular free calcium [Ca
2 þ
]
i
may cause direct
calcium-mediated mitochondrial and/or lysosomal da-
mage or activate Bax via calpain-mediated cleavage
(Mattson et al., 2000; Choi et al., 2001; Leist and
Ja
¨
a
¨
ttela
¨
, 2001). Calpains may also convert Bcl-X
L
to a
proapoptotic MMP-inducing protein (Nakagawa and
Yuan, 2000).
Calpains are cysteine proteases that reside in the
cytosol in an inactive form, and their activation requires
an elevation in [Ca
2 þ
]
i
(Wang, 2000). Calpain activity is
controlled by calpastatin, a natural inhibitor that can be
inactivated by calpain- or caspase-mediated cleavage.
Calpains are activated by various stimuli (e.g. irradia-
tion, etoposide, neurotoxins and ionophores) that
increase the [Ca
2 þ
]
i
and they can in turn participate in
PCD signalling upstream or downstream of caspases
(Leist and Ja
¨
a
¨
ttela
¨
, 2001). Furthermore, calpains
mediate apoptosis-like PCD even in the absence of
caspase activation. For example, EB1089, a vitamin D
analogue currently in phase III clinical trials for the
treatment of cancer (Mathiasen et al., 1999; Mathiasen
et al., 2002) as well as re-expression of a tumor
suppressor gene ARHI (Bao et al., 2002) induce
calpain-dependent apoptosis-like PCD in cancer cells
without triggering detectable caspase activation.
Cytoskeleton and cytosol
The cytoskeleton controls MMP by binding to ‘BH3-
only’ proteins Bim and Bmf and keeping them thereby
inactive (Coultas and Strasser, 2003). Changes in the
cytoskeleton by various cytoskeleton disrupting or
stabilizing drugs or by cell detachment can thus lead
to MMP and apoptosis induced by the release of Bim
and Bmf. On the contrary, intact cytoskeleton is needed
for autophagy to occur (Ogier-Denis and Codogno, 2003).
Tumour cells are often challenged by hypoxia and
acidosis. Hypoxia-induced factor 1a (HIF-1a) upregu-
lates the autophagy-inducing Bcl-2 protein BNIP3
(Kothari et al., 2003), whose expression level is further
increased by acidosis-mediated stabilization (Kubasiak
et al., 2002). Accordingly, BNIP3 is expressed in hypoxic
regions of tumors but fails to induce cell death
presumably due to survival signalling via tyrosine kinase
receptors (Kothari et al., 2003). Notably, HIF-a also
upregulates many genes involved in apoptosis resistance,
metabolic adaptation, angiogenesis and metastasis
making the net effect of hypoxia most likely tumour
promoting (Semenza, 2003). Furthermore, HIF-1a is
overexpressed in human cancers not only due to hypoxia
but also as a result of increased synthesis induced by
oncogenes or decreased degradation due to loss of von
Hippel–Lindau tumour suppressor protein.
Taken together, all cellular compartments appear to
be equipped with sensors that can detect stressful
changes in the environment and weapons to set off
PCD providing a cell with multiple ways to get rid of
damaged and potentially dangerous cells. Thus, tumour
cells have to get rid of many enemies on their way
through transformation and tumour progression.
Promotion and inhibition of PCD during tumourigenesis
The intensive apoptosis research during the last 15 years
has convincingly demonstrated that the development of
aggressive tumours depends on numerous defects in
apoptosis signalling (Hanahan and Weinberg, 2000).
The requirement for apoptosis resistance is partially
explained by the nature of cancer-associated growth-
promoting signals themselves. Uncontrolled prolifera-
tive responses induced by enhanced activity of onco-
proteins like c-Myc, or the inactivation of tumour
suppressor proteins like retinoblastoma protein, result
in a deregulated cell cycle and sustained activation of
E2F-1, which can trigger caspase activation and
accelerated apoptosis (Evan et al., 1992; Wu and Levine,
1994) (Table 1). Even though growth promotion and
survival functions of oncoproteins of Ras and Src
families are prominent in tumours, they also carry the
ability to trigger cellular senescence and apoptosis and
may thus suppress transformation (Yao and Scott, 1993;
Frisch and Francis, 1994; Tanaka et al., 1994; Chen
et al., 1997). Many of the stresses that a cell must
encounter on its way to form an aggressive tumour
(hypoxia, starvation, cytotoxic lymphocytes, DNA
damage as well as detachment from the extracellular
matrix and neighbouring cells) further add to the
selective pressure to suppress apoptosis. To obtain net
growth, growth-stimulating signals and environmental
stresses must thus be coupled to reduced apoptosis
potential. In line with this, human tumours harbour
several inactivating mutations in proapoptotic genes
and/or show increased expression or activity of anti-
apoptotic proteins (Table 1). For example, mutations in
proapoptotic p53 tumour suppressor gene or changes in
the expression levels of its regulators are the most
frequent genetic abnormalities in human cancers (Vous-
den and Lu, 2002). Other common antiapoptotic
alterations in cancer cells include tilted balance in the
expression levels of pro- and antiapoptotic Bcl-2 family
members towards the favour of the latter; growth factor-
independent activation of receptor tyrosine kinases; lack
of PTEN, the negative regulator of phosphatidylinositol
3-kinase (PI3K) and protein kinase B (PKB, also called
Akt); activating mutations of PKB; inactivation of
Apaf-1 in therapy-resistant melanomas; overexpression
of survivin or other inhibitor of apoptosis protein family
members; and increased levels of anti-apoptotic heat-
shock proteins (Evan and Vousden, 2001; Reed, 2001;
Simpson and Parsons, 2001; Mathiasen and Ja
¨
a
¨
ttela
¨
,
2002; Coultas and Strasser, 2003; Downward, 2003).
Emerging evidence suggest that apoptosis is not the
only lethal challenge tumour cells encounter during
tumorigenesis. Transformation and tumour progression
are also coupled to alternative death pathways that have
to be suppressed for cancer to develop and evolve
(Table 1). For example, activating mutations of ras
proto-oncogenes, the most frequently mutated proto-
oncogenes in human tumors, trigger autophagy in
glioblastoma as well as colon and gastric cancer cells
(Chi et al., 1999; Pattingre et al., 2003). The ability of
Ras to cause autophagy may explain the autophagy
Cell death pathways as regulators of tumour initiation and progression
MJa¨a¨ttela¨
2752
Oncogene
observed in prostate cancer cells following activation of
ErbB2 and ErbB3 (receptor tyroisine kinases that
activate Ras pathway) (Tal-Or et al., 2003). Interest-
ingly, one of the signalling pathways regulated by Ras,
the Raf–Erk1/2 pathway, triggers autophagy (Pattingre
et al., 2003), whereas another downstream arm of Ras
signalling, PI3K–PKB pathway, inhibits it (Arico et al.,
2001). Additionally, the Ras-induced increase in the
expression and trafficking of lysosomal cathepsins may
contribute to Ras-induced autophagy (Roshy et al.,
2003). Cells that fail to escape autophagy by activation
of PI3K–PKB pathway, may do so by inactivating
mutations or depletions of genes encoding for Beclin-1,
Bin-1 or death-associated protein kinase (DAPK), all
proteins with tumour suppressor properties (Liang et al.,
1999; Elliott et al., 2000; Ogier-Denis and Codogno,
2003). Beclin-1 is a Bcl-2-interacting protein that has
structural similarity to the yeast autophagy gene apg6/
vps30. It is monoallelically deleted in 40–75% of
sporadic human breast cancers and ovarian cancers.
Bin1, on the other hand, interacts with c-Myc and
inhibits its transforming activity. It is frequently missing
or functionally inactivated in breast and prostate
cancers and in melanoma. DAPK is a calcium-regulated
serine/threonine kinase that in addition to activating the
p19ARF/p53-mediated apoptotic checkpoint, is an
important mediator of autophagy induced by starva-
tion, antioestrogens, interferon g and TNF (Raveh et al.,
2001; Inbal et al., 2002). Human tumour cell lines and
tumours of various origins have frequently lost DAPK
expression as a result of silencing by DNA methylation
(Raveh and Kimchi, 2001). Importantly, reintroduction
of Beclin-1, Bin-1 or an active form of DAPK into
tumour cells that either lack them or express them in low
levels triggers autophagic degeneration. Furthermore,
Bcl-2, the major oncogene controlling apoptosis, can
Table 1 Examples of cancer-related events that regulate PCD
Event Death pathway Effector Effect Reference
Activation of cancer-related proteins
Bcl-2 Apoptosis Caspases Inhibition Vaux et al. (1988)
Autophagy ND Inhibition Saeki et al. (2000)
Apoptosis-like Calcium Inhibition Lam et al. (1994)
c-Myc Apoptosis Caspases Sensitization Evan et al. (1992)
Apoptosis-like AIF Sensitization Liao and Dickson (2003)
E2F-1 Apoptosis Caspases Activation Wu and Levine (1994)
Src family Apoptosis Caspases Inhibition Frisch and Francis (1994)
Activation Yao and Scott (1993)
Apoptosis-like Cathepsins Sensitization Unpublished
Ras family Apoptosis Caspases Inhibition Frisch and Francis (1994)
Activation Tanaka et al. (1994)
Autophagy ND Activation Chi et al. (1999)
Apoptosis-like Cathepsins Sensitization Unpublished
NF-kB Apoptosis Caspases Inhibition Wang et al. (1996)
Apoptosis-like Cathepsins Inhibition Liu et al. (2003)
Raf–ERK1/2 Apoptosis Caspases Inhibition Kinoshita et al. (1997)
Autophagy ND Activation Pattingre et al. (2003)
PI3K–PKB Apoptosis Caspases Inhibition Kinoshita et al. (1997)
Autophagy ND Inhibition Arico et al. (2001)
Apoptosis-like Cathepsins Inhibition Madge et al. (2003)
Hsp70 Apoptosis Caspases Sensitization Liossis et al. (1997)
Apoptosis-like Cathepsins Inhibition Unpublished
Necrosis-like ROS Inhibition Creagh et al. (2000)
Cystatin A Apoptosis-like Cathepsins Inhibition Kuopio et al. (1998)
SV40 large T Autophagy ND Inhibition Elliott et al. (2000)
Cathepsins Apoptosis-like Cathepsins Sensitization Foghsgaard et al. (2001)
ErbB2/3 Autophagy ND Activation Tal-Or et al. (2003)
Loss of function of tumour suppressor-like proteins
p53 Apoptosis Caspases Inhibition Yonish-Rouach et al. (1991)
Apoptosis-like Cathepsins Inhibition Yuan et al. (2002)
RB Apoptosis Caspases Activation Wu and Levine (1994)
PTEN Apoptosis Caspases Inhibition Stambolic et al. (1998)
Autophagy ND Inhibition Arico et al. (2001)
Beclin-1 Autophagy ND Inhibition Liang et al. (1999)
PML Apoptosis Caspases Inhibition Wang et al. (1998)
Necrosis-like ND Inhibition Quignon et al. (1998)
Bin-1 Autophagy ND Inhibition Elliott et al. (2000)
DAPK Apoptosis Caspases Inhibition Raveh et al. (2001)
Apoptosis Caspases Activation Jin and Gallagher (2003)
Autophagy ND Inhibition Inbal et al. (2002)
ARHI Apoptosis-like Calpains Inhibition Bao et al. (2002)
JNK Apoptosis Caspases Inhibition Kennedy et al. (2003)
p27
KIP
Autophagy ND Inhibition Komata et al. (2003)
Cell death pathways as regulators of tumour initiation and progression
MJa¨a¨ttela¨
2753
Oncogene
also confer protection against autophagy (Saeki et al.,
2000). Downregulation of Bcl-2 in HL60 leukaemia cells
causes autophagy in a caspase- and MMP-independent
manner (Saeki et al., 2000). The cellular change
triggering autophagy in these cells is not defined, but it
could be related to the ability of Bcl-2 to control cellular
calcium homeostasis and thereby the activation of
DAPK (Lam et al., 1994). Finally, it should be noted
that autophagy does not necessarily always inhibit
tumorigenesis, but it may in fact also promote it via
its ability to protect against stresses associated with
tumour progression, that is hypoxia, starvation and
increased apoptosis (Ogier-Denis and Codogno, 2003).
Lysosomes and the control of their stability are
affected in many ways during the tumorigenesis.
Transformation by either Ras or Src upregulates the
expression of cysteine cathepsins, increases their secre-
tion and sensitizes cells to TNF-induced cathepsin B-
dependent PCD (Roshy et al., 2003) (N Fehrenbacher
and MJ, unpublished). Accordingly, tumour progres-
sion is associated with several changes that confer
resistance to the lysosomal death pathway, that is,
activation of PI3K and NF-kB, increased expression of
cathepsin inhibitors and Hsp70, as well as loss of p53
(Table 1). Hsp70 chaperone protein is commonly
overexpressed in human, tumours and its high expression
is associated with therapy resistance and poor prognosis
(Ja
¨
a
¨
ttela
¨
, 1999). Even though Hsp70 is mainly known as
a potent survival protein, it sensitizes cells to apoptosis
induced by Fas, T-cell receptor and cytotoxic lympho-
cytes suggesting that its protective function rather
covers nonapoptotic PCD pathways (Liossis et al.,
1997; Multhoff et al., 1997). Accordingly, the depletion
of Hsp70 induces a massive caspase-independent PCD
in tumour cells of various origins and experimental
tumours in mice, indicating that transformation is
coupled to the activation of an alternative Hsp70-
controlled death pathway (Nylandsted et al., 2000;
Nylandsted et al., 2002; Frese et al., 2003). This pathway
is mediated by lysosomal cysteine cathepsins that are
released into the cytosol of Hsp70-depleted tumour cells
before any signs of cell death are evident (J Nylandsted
and MJ, unpublished). Interestingly, Hsp70 is localized
to the lysosomal membranes of cancer cells and Hsp70
positive lysosomes display increased resistance against
chemical and physical membrane destabilization.
Furthermore, Hsp70 expression in diverse cell types
effectively inhibits TNF- and anticancer drug-induced
cell death at the level of the lysosomal leakage. Thus,
Hsp70 may promote tumorigenesis by stabilizing the
lysosomal membrane. Also, the mechanism by which
NF-kB helps cells to escape the lysosomal death was
recently uncovered (Liu et al., 2003). NF-kB induces the
expression of Spi2A, a potent inhibitor of serine
proteases and lysosomal cysteine cathepsins. Also, other
inhibitors of noncaspase proteases may confer growth
advantage to metastatic tumours that frequently express
high levels of cathepsins and other proteases capable of
degrading extracellular matrix (Duffy, 1996). For
example, the expression of Cystatin A in breast cancer
correlates with an aggressive phenotype and adverse
outcome (Kuopio et al., 1998), and tissue inhibitor of
metalloproteinases-1 rescues mammary epithelial cells
from stromelysin-1-induced PCD in vivo (Alexander
et al., 1996).
Concluding remarks
Death signalling in tumour cells appears much more
complex than originally suggested by the simple caspase
activation model, and PCD can result in multiple
morphological end points brought about by caspase-
independent mechanisms or by signalling routes utiliz-
ing caspases in combination with other effectors.
Collectively, the data indicate that various forms of
PCD are triggered by processes involved in transforma-
tion and tumour progression. Indeed, elimination of
cells that have lost functions of important tumour
suppressor proteins or bear activated oncogenes by PCD
may present the primary means by which such mutant
cells are continually removed from the body. The
participation of alternative PCD pathways together
with apoptosis in this process imposes a great challenge
to the developing tumour and may explain the relative
rarity of cancer in respect to the huge number of cell
divisions and mutations during a human life.
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