APOPTOSIS AND TUMOUR

8/7/2019 APOPTOSIS AND TUMOUR

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Review

Apoptosis regulators and their role in tumorigenesis

Martin Zo« rnig aY*, Anne-Odile Hueber b, Wiebke Baum a, Gerard Evan c

a Georg-Speyer-Haus, Paul-Ehrlich-StraM e 42^44, 60596 Frankfurt, Germanyb Institute of Signaling, Developmental Biology and Cancer Research CNRS UMR 6543, Centre A. Lacassagne, 33 Avenue Valombrose,

06189 Nice, Francec 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

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-

cytes to delete autoreactive or non-reactive cells,

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.

P I I : S 0 3 0 4 - 4 1 9 X ( 0 1 ) 0 0 0 3 1 - 2

* Corresponding author. Tel. : +49-69-63395115;

Fax: +49-69-63395297.

E-mail address: [email protected] (M. Zo« rnig).

Biochimica et Biophysica Acta 1551 (2001) F1^F37

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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.

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

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-

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

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.

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



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

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 subunitsthat then assemble as an K2L2 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




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 alsodirectly 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 thesuggestion 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

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 afurther 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




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

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-1Lproduction 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 brainin 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-




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 andcell^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

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 repeatsassociated 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 involvedin 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-




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 theirphysiological 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

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 deathstimulus 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




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 memberBid [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, bax3/3bak 3/3 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

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.




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

[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




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 TRAILreceptors 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 killingin¢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�CD43CD83

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-

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 alldeath 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-




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-terminusresembles 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-U B activation. Cleavage of RIP

by caspase-8 results in the blockage of NF-U B-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 JunN-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

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 recruitmentand 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




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 ispresently 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

upregulating CD95 or CD95L [163,164] although the

generality of this concept has been questioned

[165,166].

4.3.2. TNF receptorsTNF 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-U B [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-U B [173], the latter by interaction with the down-

stream signaling kinase NIK. NIK, in turn, activates

the IU B kinases which phosphorylate and inactivate

IU B, the endogenous cellular inhibitor of NF-U B




[174]. Substantial evidence supports the notion that

NF-U B 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-

U B-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-

U B-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-U B 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-2indicate that TRAF2 is important for regulation of

lymphocyte proliferation and survival [179,180].

They are also compromised for JNK activation,

although NF-U B activation is una¡ected.

TRAF-2, TRAF-5, and TRAF-6 interact with the

downstream kinase NF-U B-inducing kinase (Nik),

which in turn interacts with the kinases within the

IU B 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-U

B 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-

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 varioustypes 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-U B 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-




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 familymembers through its interaction with TRAF pro-

teins. When so associated, TRIP inhibits TRAF-

mediated activation of the apoptosis suppressor

NF-U B. 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-U B activation by TNF-R1), TNF-R2 triggers

cell death in T-cells whereas in the absence of RIP,

TNF-R2 activates NF-U B [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

(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 `decoyreceptors' 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




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 wellas 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) triggersthe 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-

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 partbecause 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 beenidenti¢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




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 beenshown 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-

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




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 lymphocytesin 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- andT-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

(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

CD43CD83 (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-U B [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-




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, orwhether 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

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 Baxprotein 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 bak 3a3 mice were found to be develop-

mentally normal while bax3a3bak 3a3 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




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 inbax3a3 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 membraneprotein. 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

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 toundergo 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 notionreinforced 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




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 proteinP53-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

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 itsactivation 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 àpoptosome' 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 ispart 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 àpoptosome' ^ 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-




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:

bax3a3bak 3a3 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 àctive' 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-

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]. P21WAF1aCIPÀ1 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




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 p533a3 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

[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 novelgene, 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 RNAsynthesis.

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-




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 complexand 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

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 whymany 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




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.




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 studyshowed 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

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 E W

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-




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 acutemyeloblastic 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 fordeath 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].

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 apoptosiswhile 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




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-U B. 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-

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 actswith 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-




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]. Thisoncogenic 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-

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.




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