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Biochemical and Biophysical Research Communications 266, 699 –717 (1999)
Article ID bbrc.1999.1888, available online at http://www.idealibrary.com on
Apoptosis in Human Disease: A New Skin
for the Old Ceremony?
Bengt Fadeel, Sten Orrenius, 1 and Boris Zhivotovsky
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden
Received October 5, 1999
Naturally occurring cell death or apoptosis is essential for the maintenance of tissue homeostasis and
serves to remove extraneous or dangerous cells in a
swift and unobtrusive manner. Recent studies have
indicated a role for apoptosis in a plethora of human
diseases. Hence, dysregulation of apoptosis has been
implicated in autoimmune disease, acquired immune
deficiency syndrome, and other viral (and bacterial)
infections, as well as in neurodegenerative disorders
and cancer. Furthermore, dysregulated apoptosis signaling may impinge on other age-related disorders
such as osteoporosis and atherosclerosis and perhaps
on the process of aging itself. The present review provides an overview of human diseases, which are associated with defective or inadvertent apoptosis, with
examples of pathological conditions in which putative
apoptosis defects have been elucidated at the molecular level. Novel apoptosis-modulating therapeutic
strategies are also discussed. © 1999 Academic Press
Key Words: apoptosis; disease; pathogenesis;
therapy.
Life and death are inextricably entwined. Glücksmann (1), in his review of cell death in vertebrate
ontogeny almost fifty years ago, cites numerous examAbbreviations used: AICD, activation-induced cell death; AIDS,
acquired immune deficiency syndrome; AIF, apoptosis-inducing factor; ALPS, autoimmune lymphoproliferative syndrome; APAF, apoptotic protease activating factor; ARC, apoptosis repressor with
caspase recruitment domain; BH, Bcl-2 homology; ced, cell death
abnormal; egl, egg laying defective; FADD, Fas-associated death
domain-containing protein; FLICE, FADD-like ICE; FLIP, FLICEinhibitory protein; gld, generalized lymphoproliferative disorder;
HIV, human immunodeficiency virus; IAP, inhibitor of apoptosis
protein; ICE, interleukin-1b converting enzyme; IDDM, insulindependent diabetes mellitus; lpr, lymphoproliferation; MS, multiple
sclerosis; NAIP, neuronal apoptosis inhibitory protein; NF-kB, nuclear factor kB; PS, presenilin; SLE, systemic lupus erythematosus;
SMA; spinal muscular atrophy; TNF, tumor necrosis factor; TRAIL,
TNF-related apoptosis-inducing ligand; zVAD-fmk, z-Val-Ala-Aspfluoromethylketone.
1
To whom correspondence should be addressed. Fax: 146 8 32 90
41. E-mail: [email protected].
ples of naturally occurring cell death and emphasizes
its role in the sculpting of tissues and organs. Similarly, Saunders (2), in his classical study on cell death
in embryonic systems concludes that “abundant death,
often cataclysmic in its onslaught, is a part of early
development in many animals.” Yet, the study of cell
proliferation and differentiation long prevailed over
the study of cell death. In fact, naturally occurring cell
death did not receive widespread recognition until the
publication of the seminal paper on apoptosis by Kerr
and colleagues (3). These authors highlighted the significance of controlled cell deletion and described the
morphological features of this active and inherently
programmed phenomenon, which they proposed plays
a “complementary but opposite role to mitosis in the
regulation of animal cell populations” (3). The importance of their study lies not only in the detailed morphological description of apoptosis (a term derived
from the Greek word describing the falling off of petals
from a flower or leaves from a tree), but also in the
recognition of natural cell death as a wide-spread phenomenon not restricted to embryogenesis. An important corollary is that apoptosis is a genetically regulated process and as such may be amenable to
therapeutic intervention. The aim of the present review is to discuss the potential involvement of apoptosis in human disease along with novel apoptosis-based
therapeutic modalities now at hand.
THE ANATOMY OF CELL DEATH:
APOPTOSIS VS NECROSIS
Apoptosis occurs in a well-choreographed sequence
of morphological events (4). The cell first undergoes
nuclear and cytoplasmic condensation with blebbing of
the plasma membrane, a veritable dance macabre,
which has been likened to “boiling” of the cytoplasm.
Eventually, the cell breaks up into membrane-bound
fragments termed apoptotic bodies containing structurally intact organelles, as well as portions of the
nucleus. Subsequently, the apoptotic bodies are rapidly
recognized, ingested and degraded by neighboring
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cells. Apoptosis is often remarkably inconspicuous histologically, and for this reason the phenomenon as such
may have been neglected in earlier studies. Since apoptosis typically does not induce bystander cell death,
inflammation or tissue scarring, it is well suited for a
role in normal cell turnover during embryogenesis and
in adult tissues (4). Necrosis, on the other hand, is a
pathological or accidental mode of cell death, characterized by irreversible swelling of the cytoplasm and
organelles, including the mitochondria. Eventually
there is loss of membrane integrity resulting in cell
lysis and release of noxious cellular constituents. Typically, a number of contiguous cells are affected, and
exudative inflammation develops in the surrounding
tissue. Necrosis occurs when cells are subjected to toxic
stimuli such as hyperthermia, hypoxia, ischemia, complement attack, metabolic poisons and direct cell
trauma (4). Moreover, under some pathological conditions both types of cell death, i.e., necrosis and apoptosis, may occur. For instance, ischemic damage is frequently characterized by a core of acutely damaged
necrotic cells and a penumbra of cells which undergo
delayed apoptotic death (5). The “decision” of the cell to
die by necrosis or apoptosis is thought to depend
largely on the severity of the insult (6, 7).
THE APOPTOSIS MACHINERY
AND ITS REGULATION
The number of publications on apoptosis has grown
exponentially over the last decade or so, due largely to
the realization that apoptosis—the phenotype of cellular suicide—is an inherently gene-regulated process,
the core components of which are conserved through
evolution. The strongest evidence for an intrinsic death
program comes from genetic studies in the nematode
Caenorhabditis elegans (8). During postembryonic development of C. elegans, 131 of the 1090 cells die in
every nematode. Loss-of-function mutations in two
genes, ced-3 and ced-4, and gain-of-function mutations
in a third gene, ced-9 (ced, cell death abnormal), were
found to prevent all of these 131 naturally occurring
deaths. The subsequent isolation and molecular characterization of these nematode “killer” genes revealed
that ced-3, ced-4 and ced-9 all have mammalian counterparts known to be involved in apoptosis (for review
see 8). Hence, ced-3 is highly homologous to the human
interleukin-1b-converting enzyme, ICE (or caspase-1),
while ced-9 was shown to be a functional homologue of
the human anti-apoptotic protein Bcl-2. More recently,
APAF-1 (apoptotic protease-activating factor-1) was
demonstrated to be a human homologue of ced-4 (9)
and additional mammalian ced-4 homologues have
since then been unveiled (10). Collectively, these findings have underscored the remarkable degree of conservation of the cell death pathway from nematodes to
humans, and are suggestive of the existence of a core
death machinery in all cells.
A detailed account of the apoptosis signaling pathways employed in mammalian cells is beyond the scope
of the present discussion, and has been subject to excellent reviews elsewhere (11, 12). However, it is important to note that the cytoplasmic cysteine proteases
known as caspases, now comprising a family of 14
mammalian members, have emerged as the central
executioners of apoptosis elicited by a variety of apoptotic stimuli (Fig. 1). In addition to their role in energy
production, mitochondria have been also suggested to
be critically involved in apoptosis signaling, with the
early dissipation of mitochondrial transmembrane potential serving as a harbinger of death in numerous
systems. Mitochondria are known to release apoptogenic factors upon apoptosis triggering, including the
recently characterized AIF (apoptosis-inducing factor),
cytochrome c and several pro-caspases, earning these
organelles the epithet “poison cupboard” of the cell
(13). The plasma membrane receptor designated Fas
(also known as APO-1 or CD95) has emerged as a key
regulator of apoptosis within the immune system. Fasmediated apoptosis transpires via a caspase-dependent
pathway, with mitochondria serving in some cases to
amplify the initial death signal. The importance of Fas
and its corresponding ligand in the homeostatic regulation of immune responses was underscored by the
abnormalities observed in mice homozygous for the lpr
and gld loci (14). As a result of mutations in the Fas
and Fas ligand gene, respectively, these mice develop
profound autoimmune disease, apparently due to the
lack of apoptotic deletion of autoreactive lymphocytes.
Apoptosis signaling instigated through death receptors
such as Fas or directly via mitochondria is regulated,
in turn, by the pro- and anti-apoptotic members of the
rapidly expanding Bcl-2 family, which includes several
human homologues of the recently characterized C.
elegans death activator egl-1 (8, 15). Finally, the swift
recognition and engulfment of moribund cells by neighboring phagocytes is an important part of the apoptotic
process (16). Indeed, apoptosis can be viewed essentially as a convenient cell clearance mechanism. Numerous candidate receptors and ligands have been
identified which regulate phagocytosis of apoptotic
cells in mammalian systems (17). In addition, genes
regulating the clandestine clearance of dead cells in C.
elegans have also been characterized and have in some
cases proven to be homologues of known mammalian
genes (8).
APOPTOSIS IN HUMAN DISEASE:
TOO MUCH OR TOO LITTLE
In every human being about a hundred thousand
cells are produced every second by mitosis, and a sim-
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FIG. 1. The road to death: A current model of apoptosis signaling.
ilar number die by apoptosis (12). It is therefore of
paramount importance that these events be tightly
regulated. Consequently, the malfunction of the death
machinery intrinsic to every cell may play a primary or
secondary role in various diseases, with essentially too
little or too much apoptosis (or apoptosis occurring in
the wrong place and/or at the wrong time) leading to
proliferative or degenerative diseases, respectively
(Table 1). In the following sections some illustrative
examples of diseases where apoptosis dysregulation
has been implicated are highlighted, together with a
brief discussion of the potential apoptosis-modulating
therapeutic modalities prompted by these findings. Of
note, evidence has now accrued for a role of apoptosis
in most of the major human pathologies, including
cancer, stroke and ischemic heart disease, and putative
apoptotic mechanisms in disease should therefore not
be merely of academic interest. However, the involvement of apoptosis in disease is not always straightforward, as demonstrated by the fact that certain viral
infections trigger apoptosis while others appear to retard this process (Table 1). Moreover, autoimmunity
may arise from a lack of apoptotic cell deletion as
discussed earlier for the lpr and gld mice, although in
some cases, unscheduled apoptotic destruction of cells
may, instead, contribute to organ failure and autoimmunity. Finally, while cancer may be viewed simply as
a result of ineffective apoptosis and hence a net gain of
cells due to unrestrained proliferation, cancer cells
themselves may evade immune surveillance by triggering apoptosis of patrolling immune cells. The following
discussion aims to clarify these issues, and while in the
process of doing so, perhaps give rise to some new and
unanswered questions.
AUTOIMMUNE DISEASE
Systemic autoimmunity. The activation of T lymphocytes is known to lead to the concomitant upregulation of Fas and Fas ligand and to the susceptibility of
these cells to Fas-mediated killing (so-called AICD, or
activation-induced cell death) (18). AICD is essentially
a mechanism for “switching off” the immune response
and serves to limit the intensity of immune responses
that might otherwise prove debilitating. It has been
argued that chronic inflammatory disease, such as
asthma, could result from an escape of activated T
lymphocytes from Fas/Fas ligand-mediated cell death
(19). Zhou et al. (20) showed that the protein kinase C
inhibitor, bisindolylmaleimide VIII, markedly enhances the sensitivity of T lymphocytes to AICD.
Treatment of animals with bisindolylmaleimide VIII in
vivo abrogated autoantigen-specific immune responses
as well as autoimmunity in two different models of T
lymphocyte-mediated autoimmune disease. This indicates that accelerated induction of apoptosis in T lymphocytes can limit autoantigen-driven immune responses and thus offers a novel strategy for the
treatment of autoimmune disease.
Studies conducted in lpr and gld mice have elucidated the role of the Fas system in peripheral selection
and the maintenance of immune tolerance (14). Importantly, a syndrome in humans which closely resembles
the murine phenotype has recently been described.
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TABLE 1
To Die or Not to Die: Role of Apoptosis in Human Disease
Diseases associated with increased apoptosis
Diseases associated with decreased apoptosis
1. Neurodegenerative disorders
Alzheimer’s disease
Amyotrophic lateral sclerosis
Creutzfeld–Jakob’s disease
Huntington’s disease
Parkinson’s disease
Retinitis pigmentosa
Spinal muscular atrophy
2. Hematological disorders
Aplastic anemia
Fanconi anemia
Hodgkin’s disease
Myelodysplastic syndromes
Polycythemia vera
3. Autoimmune disorders
Fulminant hepatitis
Graft-versus-host disease
Hashimoto’s thyroiditis
Insulin-dependent diabetes mellitus
Multiple sclerosis
Rheumathoid arthritis
Scleroderma
Sjögren’s syndrome
4. Ischemic injury
Ischemia and reperfusion
Kidney infarction
Myocardial infarction
Stroke
5. Toxin-induced disease
Alcohol-induced hepatitis
Pulmonary fibrosis
Sepsis
6. Bacterial and viral infection
Acquired immune deficiency syndrome
Ebola virus
Chlamydia trachomatis
Helicobacter pylori
Neisseria meningitidis
Salmonella typhimurium
Shigella flexneri
7. Miscellaneous
Traumatic spinal cord injury
Tumor counterattack (immune privilege)
1. Cancer
Blastoma
Carcinoma
Leukemia
Lymphoma
Malignant glioma
Sarcoma
Seminoma
2. Premalignant disease
Ataxia telangiectasia
Paroxysmal nocturnal hemoglobinuria
Myelodysplastic syndromes
Xeroderma pigmentosum
3. Autoimmune disorders
Autoimmune lymphoproliferative syndrome (types I and II)
Systemic lupus erythematosus
4. Atherosclerosis
5. Metabolic disorders
Niemann–Pick’s disease
Osteoporosis
Wilson’s disease
6. Viral infections
Adenoviruses
Baculoviruses
Epstein–Barr virus
Herpesviruses
Poxviruses
Apoptosis in physiological and premature aging
Down’s syndrome
Progeria
Xeroderma pigmentosum
Two children were initially reported who displayed
progressive lymphoproliferation associated with autoimmunity and large numbers of “double negative” T
lymphocytes in the peripheral lymphoid system. Cellular and molecular studies of unrelated children with
similar clinical findings revealed profound defects in
lymphocyte apoptosis associated with mutations in Fas
(21, 22). These children were grouped into a single
entity designated autoimmune lymphoproliferative
syndrome (ALPS). Additional reports have surfaced
since the original characterization of ALPS, including a
study of patients with Canale-Smith syndrome, which
was interpreted as being identical to ALPS (23, 24).
Moreover, two kindreds with a related, though more
severe, phenotype were recently described (25). This
syndrome, termed ALPS type II, is characterized by
defective lymphocyte and dendritic cell apoptosis, yet
with no molecular abnormalities in Fas or Fas ligand.
Instead, these patients harbored mutations in
caspase-10 resulting in impaired Fas-mediated apoptosis in lymphocytes as well as defective TRAIL-induced
killing in both lymphocytes and dendritic cells.
Systemic lupus erythematosus (SLE) is a prototype
systemic autoimmune disease, which is characterized
by the presence of multiple autoantibodies and multiorgan immune destruction. A role for the Fas system in
SLE was suggested by studies on soluble Fas (sFas),
i.e., secreted Fas molecules derived from alternative
splicing of Fas transcripts. Mice injected with sFas
develop autoimmune symptoms and elevated serum
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levels of sFas were found in a number of SLE patients
(26). Based on these findings, it was suggested that
sFas may act as a ligand-binding decoy, thus preventing the apoptotic deletion of potentially autoreactive
lymphocytes. Moreover, screening of DNA from 75 SLE
patients has identified one individual with a heterozygous Fas ligand gene mutation with concomitant defects in Fas ligand function and AICD (27). CasciolaRosen et al. (28) have shown that nuclear
autoantigens, such as those targeted in SLE, are redistributed into cell surface blebs of apoptotic cells, and it
was concluded that these surface blebs might serve as
a stimulus for autoantibody production. Macrophage
engulfment of apoptotic cells in vitro was decreased in
SLE patients, and it was suggested that persistently
circulating “apoptotic waste” (i.e., cells which are not
effectively phagocytosed) may serve as immunogen for
the induction of autoreactive lymphocytes in vivo in
these patients (29). In addition, the observation of increased numbers of uncleared apoptotic cells in the
kidneys of the SLE-susceptible C1q-null mouse
strengthens the idea that abnormal clearance of apoptotic cells may play a role in the pathogenesis of SLE
(30). Taken together, these findings suggest that the
defective clearance of apoptotic cells may represent a
hitherto unrecognized role in the initiation and progression of autoimmune disease (16).
Organ-specific autoimmunity. In contrast to systemic autoimmune disorders that are characterized by
B lymphocyte stimulation leading to antibody formation and autoimmune complex-mediated tissue injury,
organ-specific diseases are characterized by T
lymphocyte-mediated attack on specific cell types
within the organ (31). Cell targets include the b-cells of
the islets of Langerhans in insulin-dependent diabetes
mellitus (IDDM), oligodendrocytes in the brain in multiple sclerosis (MS) and thyrocytes in Hashimoto’s thyroiditis. Recent data have implicated the Fas/Fas ligand system in the destructive phase of these
autoimmune disorders (31). The first-described organspecific autoimmune disease was Hashimoto’s thyroiditis (HT), yet attempts to define the mechanism responsible for thyrocyte destruction were unsuccessful for
many years. Kotani et al. (32) provided evidence for
apoptotic death in the thyroid gland of patients with
HT. Thyrocytes from HT glands expressed Fas
whereas non-autoimmune thyroids exhibited negligible amounts of Fas (33). On the other hand, Fas ligand
was constitutively expressed in both normal and HT
thyrocytes. It is likely that an autocrine selfdestruction of thyrocytes, which coexpress Fas and Fas
ligand, is a type of cell death analogous to AICD in T
lymphocytes. Finally, recent data indicate that the Fas
ligand-related TRAIL death pathway might also be
operational in thyroid disease (34).
Multiple sclerosis (MS) is a demyelinating disease of
the central nervous system in which myelin and
myelin-producing oligodendrocytes become the target
of an inflammatory process resulting in plaque formation and neurological symptoms. Since destruction of
oligodendrocytes results in the loss of myelin and a
concomitant impairment of nerve conduction, considerable effort has been devoted to the identification of
the mechanism(s) responsible for the death of these
cells. In normal white matter, Fas expression is weak
and scarce. However, analysis of acute and chronic MS
plaques revealed a diffuse and intense Fas reactivity
among the oligodendrocytes located along the lesion
margin and in the adjacent white matter, along with
the occurrence of Fas ligand-expressing microglia and
infiltrating lymphocytes (35). In addition, in vitro studies have shown that oligodendrocytes are susceptible to
Fas-mediated killing (35), and that Fas governs astrocyte apoptotic responses in vitro (36), which might be of
potential importance in MS. These observations are
compatible with a role for Fas and Fas ligand in the
destruction of oligodendrocytes in MS patients.
Insulin-dependent diabetes mellitus (IDDM) is a
chronic autoimmune disease resulting from the destruction of insulin-producing b-cells in the pancreas.
Much of what is known about the pathogenesis of
IDDM comes from studies of the nonobese diabetic
(NOD) mouse. Itoh et al. (37) showed that NOD mice
bearing the lpr mutation were protected from the development of diabetes and adoptive transfer of splenocytes from diabetic NOD to these animals also failed to
induce disease, suggesting that the Fas/Fas ligand system plays a key effector role in the pathogenesis of
IDDM. On the other hand, Allison and Strasser (38)
reported that Fas plays only a minor role in b-cell
death in diabetic mice. Calcium is one of the most
versatile and universal signaling molecules in the body
and can function as either an inducer or inhibitor of
apoptosis (39). We previously showed that specific calcium channel blockers prevent apoptosis in pancreatic
b-cells treated with serum from patients with type I
diabetes (40). Similarly, Srinivasan et al. (41) have
shown that serum from type II diabetic patients with
neuropathy induces calcium-dependent neuronal apoptosis. Moreover, high glucose concentrations and tolbutamide induce apoptosis in b-cells, which is dependent
on intracellular calcium (42). Collectively, these findings have implications for the autoimmune destruction
of insulin-producing b-cells evidenced in diabetes patients.
NEURODEGENERATIVE DISEASE
In the vertebrate nervous system up to 50% or more
of different types of neurons die before embryonic development is complete. In other words, death gives
birth to the nervous system. Only those neurons receiv-
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ing enough neurotrophic support from their target cells
will survive, and the rest are believed to be weeded out
by the process of apoptosis (43). It has become apparent
that excessive or inadvertent apoptosis also occurs in
various pathological conditions in the brain (44). Particular interest has been devoted to the role of caspases
in the pathogenesis of chronic neurodegenerative conditions such as Alzheimer’s disease and Huntington’s
disease, although a role for caspases in acute traumatic
neuronal injury has also been suggested (45). In addition, a promising caspase-based approach for the amelioration of nigral tissue transplant survival was recently reported (46). In this study, which may have
important implications for the treatment of Parkinson’s disease, caspase inhibitors were shown to reduce
apoptosis in embryonic nigral cell suspensions and to
improve survival of dopaminergic neurons grafted to
hemiparkinsonian rats, thereby improving functional
recovery in these animals. The importance of caspases
in neuronal apoptosis was further underscored by the
restricted phenotype of caspase-3 and caspase-9 knockout animals, which displayed profound abnormalities
in neural tissues despite a normal development of
other organs including lung, liver and thymus (47).
Alzheimer’s disease. Alzheimer’s disease (AD) is
the most common form of adult-onset dementia. AD is
characterized by distinct neuropathological lesions including intracellular neurofibrillary tangle formation,
extracellular deposition of b-amyloid, loss of synapses
and neurodegeneration. The b-amyloid peptide has
been shown to induce apoptosis in cultured neurons
(48), and recent data indicate that the b-amyloid precursor protein may serve as a caspase substrate (49),
thereby directly incriminating caspases in amyloidogenesis in AD. Genes encoding presenilin-1 (PS1) and
presenilin-2 (PS2) were found to be mutated in most
cases of early-onset familial Alzheimer’s disease (50).
The cellular consequences of these mutations include
an aberrant accumulation of b-amyloid peptide, derived from the b-amyloid precursor protein. Two
apoptosis-promoting proteins, p53 and p21 waf-1, both
inhibit PS1 production, suggesting that presenilins are
normally anti-apoptotic. In fact, the presenilins are not
only “switched off” during apoptosis, they can also
undergo proteolytic processing associated with the activation of caspases (51). Such caspase-dependent
cleavage results in loss of their anti-apoptotic function.
Buxbaum et al. (52) have reported a novel calciumbinding protein, calsenilin, that binds both PS1 and
PS2 and regulates the levels of their proteolytic products, thus providing a possible link between the presenilins, calcium homeostasis and caspase activation.
Calcium presumably serves to induce conformational
changes in calsenilin, with subsequent changes in the
levels of anti-apoptotic presenilins. Moreover, ALG-3
(apoptosis-linked gene-3), a truncated PS2 cDNA en-
coding the calsenilin-binding region of PS2, protects
against apoptosis possibly by sequestering calsenilin
away from PS1 and PS2 (53). In sum, it is likely that
b-amyloid peptide is the causative agent in Alzheimer’s
disease, and PS mutations, along with activation of
neuronal caspases, may act to increase the levels of
this pathological peptide, thus leading to an increase in
the degree of apoptotic destruction of neurons.
Spinal muscular atrophy. The childhood spinal
muscular atrophies (SMAs), characterized by spinal
cord motor neuron depletion, are among the most common autosomal recessive disorders. SMAs are subclassified as either type I, type II or type III, according to
the age of onset and clinical severity, and the disease
manifests as weakness and wasting of the voluntary
muscles. Type I SMA is the most severe form and
affected children rarely survive the first few years owing to respiratory muscle weakness. Many of the motor
neurons observed at autopsy in the spinal cord of these
patients are characterized by swelling and chromatolysis in a manner consistent with an apoptotic mode of
cell death (54). The gene for neuronal apoptosis inhibitory protein (NAIP), a novel baculovirus IAP homologue, was recently mapped to the SMA region of chromosome 5q13.1 and found to be deleted in type I SMA
individuals (55). These findings suggested that NAIP
acts to promote neuronal survival, and that SMA
might be caused by the pathological persistence or
reactivation of normally occurring apoptosis in the spinal cord due to a lack of NAIP activity. Interestingly,
the elevation of neuronal NAIP expression also confers
resistance to ischemic damage in animal models of
stroke (56). Several additional IAP homologues have
been identified, including the four human IAPs,
cIAP-1, cIAP-2, XIAP and survivin (57). The mechanism by which these novel proteins block cell death is
largely unclear, but appears to depend, at least in some
cases, on the direct inhibition of caspases-3 and -7 and
of cytochrome c-induced activation of caspase-9 (58).
Amyotrophic lateral sclerosis. Amyotrophic lateral
sclerosis (ALS) is an adult-onset neurodegenerative
disease that occurs in both sporadic and familial forms.
About 20% of the familial forms are associated with
mutations in the gene encoding the cytosolic Cu/Zn
superoxide dismutase (SOD) (59). Transgenic mice
which express human familial ALS-associated mutant
SOD develop a motor neuron degenerative syndrome
that closely resembles ALS (60). Interestingly, the wild
type Cu/Zn SOD acts as an anti-apoptotic gene in cultured neural cells, whereas the familial ALSassociated mutants act as dominant proapoptotic
genes, despite the retention of significant SOD activity
by some of the mutants (61). In situ hybridization
studies revealed a decreased expression of Bcl-2 and a
concomitant increase in Bax in ALS spinal cord motor
neurons (62), suggesting that apoptotic mechanisms
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may be involved in the attrition of cells in these patients. Bcl-2 overexpression was also found to delay the
onset of symptoms, but not disease progression, in a
transgenic mouse model of ALS (63). Furthermore, a
potential role for caspases in ALS was demonstrated by
the significant slowing in the progression of symptoms
following inhibition of caspase-1 in mice (64).
Huntington’s disease. HD is a progressive neurodegenerative disease, which belongs to a group of disorders including dentatorubropallidoluysian atrophy,
spinocerebellar ataxia type 3, and spinal bulbar muscular atrophy. The genetic defect in each of these diseases is the expansion of a CAG trinucleotide in the
encoding region of their respective genes, which is
translated into a polyglutamine repeat. How polyglutamine expansion culminates in selective neuronal loss
is unknown, although it has been shown that neuronal
death may occur by apoptosis in HD (65). Disruption of
the HD gene in mice caused increased neuronal apoptosis and progressive behavioral and motor dysfunction (66). The HD gene product, huntingtin, is a substrate of caspase-3, or a closely related protease, and
its cleavage was modulated by the length of the polyglutamine tract (67). Cytosolic aggregates of polyglutamine repeat proteins may recruit procaspase-8, most
likely through binding of the adaptor protein FADD,
resulting in the activation of caspase-8 (68). Taken
together, these results suggest that caspase activation
might play a role in HD, although it is presently unclear how the fragments liberated by caspase cleavage
are linked to the demise of specific neuronal populations. Nevertheless, if the relationship between toxicity
and caspase-mediated cleavage events can be formally
demonstrated, then the administration of caspase inhibitors may, theoretically, serve to mitigate against
cell death in these patients
VIRAL AND BACTERIAL INFECTION
Viral disease. Two diametrically opposed hypotheses have emerged for the role of apoptosis in viral
infections: (1) as a host antiviral defense mechanism,
versus (2) as a pathogen-mediated mechanism to induce immune dysregulation and promote persistent
infection. The validity of either of these hypotheses
appears to depend on the specific viral infection examined (69). Hence, apoptosis of an infected cell may in
some cases be viewed as a defense mechanism to prevent viral propagation, and the typical DNA fragmentation associated with apoptotic death may serve to
eliminate viral DNA sequences which have been incorporated into the cellular genome. Cytotoxic T cells also
act to prevent viral spread by recognizing and killing
virally infected cells via a Fas/Fas ligand-dependent
mechanism. To circumvent these host defenses, many
viruses have evolved exquisite strategies to inhibit
apoptosis of the infected cell. For example, establish-
ment of an effective adenoviral infection depends on
the function of the E1B 19K protein, a viral homologue
of anti-apoptotic protein Bcl-2 (70). Another adenovirus protein termed RID (receptor internalization and
degradation) mediates internalization of cell surface
Fas and subsequent destruction inside lysosomes
within the cells, which may allow cells to resist Fasmediated death and thus promote the survival of the
virus (71). The p35 protein and the inhibitor of apoptosis proteins (IAPs) found in baculoviruses can inhibit
apoptosis in response to a wide variety of stimuli (72),
and cellular IAPs have subsequently been identified
that are presumed to act as direct inhibitors of
caspases. In addition, the cowpox serpin crmA has also
been shown to inhibit the activation of caspases (73).
Numerous herpesviruses, including Kaposi’s sarcomaassociated herpesvirus (KSHV, also known as HHV-8)
and herpesvirus saimiri, contain FLIPs (FLICEinhibitory proteins) which possess sequences that interact with the Fas-associated adaptor protein, FADD
(74). This FLIP-FADD interaction is thought to inhibit
signaling events induced upon ligation of Fas or the
TNF receptor by their respective ligands (74). Conversely, other viruses trigger apoptosis and/or cell lysis, perhaps for the purpose of facilitating viral release
toward the end of the viral replicative cycle, and
thereby succeed to promote viral infectivity and pathogenesis. A vivid example is provided by recent studies
in victims of Ebola virus-infection, in which fatal outcome in patients was associated with massive intravascular apoptosis (75).
AIDS. Infection with human immunodeficiency virus is characterized by the gradual depletion of CD4 1 T
cells and the subsequent emergence of opportunistic
infections and other manifestations of immunodeficiency which are collectively termed acquired immune
deficiency syndrome (AIDS). It is therefore axiomatic
that understanding the pathogenesis of HIV infection
requires an understanding of the mechanisms involved
in the loss of CD4 1 T cells. A few years ago it was
hypothesized that programmed cell death might account for the cell dysfunction and depletion in AIDS
(76), and subsequent reports have substantiated the
enhanced propensity of cells from HIV-infected individuals to undergo apoptosis. An increased susceptibility
in both the CD4 1 and CD8 1 subpopulation of T cells
has been described (77), and this appears in some cases
to correlate with disease progression (78), although
these findings have been disputed by others (79). Furthermore, it now seems apparent that during HIV disease, the battle between host and virus spills out into
civilian territory, with the majority of T cell deaths in
vivo occurring via apoptosis in uninfected “bystander”
cells rather than in cells actually infected with the
virus (80). Several mechanisms have been proposed to
account for the enhanced degree of apoptosis induction
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evidenced in these patients, including apoptosis mediated by the HIV transactivator protein tat (81), AICD
mediated by Fas/Fas ligand interactions (82), interaction of the viral envelope glycoprotein gp120 with
CD4 1 (83) or with the chemokine receptor CXCR4 (84)
and cleavage of endogenous Bcl-2 by the HIV protease
(85). However, apoptosis during the course of HIV infection may not always be detrimental to the host. The
prevention of apoptosis in infected CD4 1 T cells by
adenovirus E1B 19K transfection resulted in enhanced
viral production and the establishment of persistent
high-level infection (86). More recently, Chinnaiyan et
al. (87) arrived at similar conclusions, in a study aimed
at the inhibition of caspases in HIV-infected cells, thus
casting doubt on the feasibility of anti-apoptotic therapeutic modalities in HIV-infected individuals. On the
other hand, a novel “Trojan horse” strategy to kill
HIV-infected cells specifically was presented (88). By
substituting proteolytic cleavage sites, these authors
engineered an apoptosis-promoting caspase-3 protein
that was processed into its active form by the HIV
protease, resulting in apoptosis of the infected cell, but
which remained inactive in uninfected cells. Future
studies will show whether such an approach is fruitful
in combating HIV infection.
Bacterial disease. An increasing number of bacteria have been identified as triggers of apoptosis in vitro
(89). Activation of apoptosis has been proposed to serve
as an efficient means for the bacterial “David” to strike
the eukaryotic “Goliath” (89). For example, macrophages undergo apoptosis in vitro upon infection with
Salmonella typhimurium, Shigella flexneri, and Yersinia pseudotuberculosis. It has been speculated that
the induction of macrophage death may be important
to initiate infection, promote bacterial survival and
escape from host immune responses. Recent data indicate that IpaB, a bacterial invasin of S. flexneri, induces apoptosis by binding directly to caspase-1, a component of the macrophage apoptosis machinery (90).
Since caspase-1 is also a pro-inflammatory molecule,
induction of apoptosis may serve not only to delete host
cells, but also to initiate inflammation. These findings
raise the possibility of modulating the interaction between IpaB and caspase-1 as a target in both therapy
and vaccine development. Moreover, a broad-spectrum
caspase inhibitor, zVAD-fmk, prevented neuronal apoptosis and meningeal inflammation in an animal model
of acute pneumococcal meningitis (91). The authors
suggested that such caspase inhibitors, in conjunction
with antibiotics, may provide a novel adjunctive treatment of bacterial meningitis and perhaps aid in the
prevention of neurologic sequelae associated with this
disease. It was reported recently that the obligate intracellular bacterial pathogen Chlamydia trachomatis,
a leading cause of many important sexually transmitted diseases worldwide, possesses a novel anti-
apoptosis mechanism, namely the blockade of mitochondrial cytochrome c release and subsequent
caspase activation (92). Again, the elucidation of specific molecular targets of invading microbial agents
may allow for more selective therapy of bacterial diseases.
The Neisseria porin protein PorB, a member of the
large family of pore-forming proteins produced by
gram-negative bacteria, is capable of translocating into
membranes of infected target cells and functions in the
infection process via an increase of cytosolic free calcium and subsequent activation of calpain as well as
caspases (93). Intriguingly, PorB shares homology with
the mitochondrial voltage-dependent anion channel
(VDAC) and was therefore suggested to be a precursor
of the permeability transition pore, a putative central
regulator of apoptosis in mammalian cells. Helicobacter pylori infection is associated with chronic gastritis,
peptic ulceration and gastric carcinoma (94). Helicobacter pylori triggers apoptosis in gastric epithelial
cells and this process involves the activation of the Fas
system (95). The involvement of Helicobacter pylori
infection in the development of duodenal ulcers in vivo
has been reported recently (96). It is possible that in
the future, treatment protocols for the eradication of
Helicobacter pylori infection may also include “antiapoptotics” in addition to conventional antibiotics.
CANCER
Apoptosis and proliferation may be viewed as terms
of the “growth equation” and too much growth, as in
the case of cancer development, may result from too
little death as well as from too much proliferation.
Indeed, recent data have established a role for apoptosis dysregulation in all facets of cancer development,
including hyperplasia, neoplastic transformation, tumor expansion, neovascularization and metastasis.
Moreover, if cancer arises due to mutations that lead to
the dysregulation of normal apoptotic death, then antineoplastic therapies designed to circumvent these intrinsic defects and specifically trigger apoptosis should
prove beneficial. Novel apoptosis-based therapeutic
tools would perhaps in the future be tailored specifically to the requirements of individual patients, depending on the profile of resistance and/or susceptibility toward apoptosis induction in ex vivo assays or on
the profile of apoptosis-related genetic aberrations in
in silico (i.e., microarray-based) assays. The molecular
basis for apoptosis dysregulation in cancer and cancer
therapy will be reviewed in the following sections.
Oncogenes and tumor suppressor genes. Wyllie et
al. (97) showed that cells which overexpress normal ras
or myc protooncogenes induce tumors with high rates
of both apoptosis and mitosis. However, cells that ex-
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pressed the mutated, oncogenic form of the ras gene
induced tumors with large numbers of mitotic cells, but
few apoptotic cells. This was the first experimental
evidence for the function of oncogenes as anti-apoptotic
genes. Depending on the availability of critical growth
factors expression of c-myc determines whether cells
will undergo continuous proliferation or apoptosis (98).
It is likely that c-myc does not itself induce apoptosis,
but rather acts to sensitize cells to a wide range of
distinct insults, such as serum or growth factor deprivation. For instance, c-myc was shown to act downstream of Fas through sensitization of cells to Fasmediated killing (99). In addition, activation of c-myc
induces sensitization to apoptosis through caspaseindependent cytochrome c release from mitochondria,
and the addition of survival factors blocks this release
(100). Thus, the fate of a cell expressing c-myc is
acutely dependent on both the availability of survival
factors and on other signaling pathways, which may
determine cell transition between growth arrest, proliferation and apoptosis.
Soon after the pioneering observations made by Wyllie and colleagues (97), Vaux et al. (101) provided the
first clues for the role of another oncogene in apoptosis.
These authors found that bcl-2, the translocation of
which results in the progression of high-grade lymphomas, extends pre-B cell survival and that it cooperates
with the myc oncogene to immortalize lymphocyte precursors. Additional studies revealed that Bcl-2 contributes to tumorigenesis by extending cell survival
through direct inhibition of apoptosis (102). Pathological elevations in the levels of one or more apoptosissuppressing proteins of the Bcl-2 family (Bcl-2, Bcl-X L,
Bcl-W, Mcl-1 and A1) have since been observed in
several types of cancer. For example, high levels of
Bcl-2 were observed in patients with B-cell chronic
lymphocytic leukemia (B-CLL), although in this disease transcriptional activation rather than chromosomal abnormalities at the bcl-2 locus appear to be
involved (103). Enhanced expression of Bcl-2 also appears to be common in neuroblastoma and is associated
with unfavorable histology (104). However, in many
tumors there may not be a direct correlation between
overexpression of Bcl-2 and disease progression. For
example, although small cell lung cancer (SCLC) cells
expressed high level of Bcl-2 protein, these cells were
more sensitive to spontaneous as well as radiationinduced apoptosis compared with non-small cell lung
cancer (NSCLC) cells, which express low level of this
protein (105). A decreased expression of Bax and Bak,
pro-apoptotic members of the Bcl-2 family, has been
reported in several human cancers (106). Moreover,
experiments with knockout animals clearly demonstrated the ability of Bax to function as a tumor suppressor in vivo (107). In addition to transcriptional
regulation of Bcl-2 family members, these molecules
are also subject to post-transcriptional regulation (15).
These mechanisms include the modification of proteins
such as Bad via phosphorylation by Akt/Protein kinase
B and other kinases. Bad is present as a doublet in
many tumor cells, consistent with the presence of hyperphosphorylated Bad protein (108). Thus, high levels
of phosphorylated Bad, along with other pro-apoptotic
proteins from the same family, may conceivably be
linked to resistance to therapy. On the other hand, it
has been shown that phosphorylation of the antiapoptotic proteins Bcl-2 and Bcl-X L leads to their inactivation and most likely to the sensitization of tumor
cells to therapy (109). Furthermore, Bcl-2 can be
cleaved by caspases in response to chemotherapeutic
agents in vitro, resulting most likely in the loss of its
anti-apoptotic function (110). Future studies will show
whether cleavage of Bcl-2 plays a role in the sensitivity
of tumor cells to treatment.
Mutations in p53 are the most common chromosomal
aberrations in human cancer. The role of apoptosis in
malignant disease was underscored by the observation
that p53 can induce apoptosis (111) and that p53dependent apoptosis can serve as a critical regulator of
tumorigenesis (for review see 112). Although p53 is
dispensable for normal development, experiments conducted in knockout mice confirmed that p53 acts as a
tumor suppressor in vivo (113). Activation of p53 by
DNA-damaging agents is critical for eliminating cells
with damaged genomic DNA and underlies the apoptotic response of human cancers treated with ionizing
radiation and DNA-damaging drugs (112). When cells
suffer DNA damage, the p53 protein, also known as
“the guardian of the genome” (114), accumulates due to
post-translational stabilization and blocks entry into S
phase. This transient delay at the G1 checkpoint in the
cell cycle permits repair of damaged DNA prior to DNA
replication. However, if DNA repair fails, p53 may
trigger apoptosis. The expression of the p21 waf-1 gene is
directly induced by p53, and the waf-1 protein has been
implicated as an important effector of p53-mediated
growth arrest (112). Using the serial analysis of gene
expression (SAGE) technique, Polyak et al. (115) examined the transcripts induced by p53 expression, and
found that many of the p53-induced genes (PIGs) encoded proteins related to the redox status of the cell.
p53 is also known to upregulate the expression of the
bax gene (116), thereby altering the apoptotic “rheostat” of the cell, i.e., the ratio of Bcl-2-to-Bax heterodimer formation. Another candidate effector molecule is the cell surface receptor Fas, which is
transcriptionally upregulated by p53 (117). A recent
report has also demonstrated that p53 can induce
apoptosis through trafficking of Fas from intracellular
stores to the cell surface (118). Moreover, APAF-1 and
caspase-9 were recently found to serve as downstream
effectors of p53-dependent apoptosis, and the disruption of these effector mechanisms of apoptosis facili-
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tated oncogenic transformation and tumor development in mice (119).
Resistance to cancer therapy. The observation that
anti-neoplastic drugs may trigger apoptotic cell death
was first reported by Searle et al. (120), yet remained
neglected until recently. Today we know that a wide
variety of chemotherapeutic agents induce apoptosis in
tumor cell lines in vitro. Specifically, recent data indicates that apoptosis induced by various chemotherapeutic agents may involve the Fas/Fas ligand system
(121). Conversely, resistance against chemotherapy
may conceivably arise due to mutations in components
of the apoptotic machinery. A flurry of recent reports
have described the occurrence of Fas mutations in patients with multiple myeloma (122), non-Hodgkin’s
lymphoma (123) and adult T cell leukemia (124). In
addition, recent studies have shown that resistance to
chemotherapy is associated with resistance to Fasmediated apoptosis (125), while others have provided
evidence that Fas-independent pathways may also be
involved (126). We have found that small cell lung
cancer (SCLC) cells are lacking in pro-caspase-1, -4, -8,
and -10 compared to non-small cell lung carcinomas
(NSCLC), although the significance of these findings is
presently unclear since SLCC cells are generally more
sensitive to chemotherapy-triggered apoptosis (127).
Similarly, the breast carcinoma cell line, MCF-7, carries a 47-bp deletion within exon 3 of the caspase-3
gene, although these cells remain susceptible to apoptosis triggering (128).
The Fas system may also play a role in the escape of
tumor cells from immune surveillance. Hence, high
constitutive Fas ligand expression has been demonstrated in different tumors and it was suggested that
such tumors may eliminate activated Fas-expressing
lymphocytes by induction of apoptosis (129). In addition, a novel Fas ligand decoy, DcR3 (decoy receptor 3)
was recently identified, and suggested to enable tumor
cells to evade surveillance by Fas ligand-expressing
cytotoxic lymphocytes (130). In a sense, the tumor can
be viewed as an immune privileged site (discussed
below). Indeed, the combination of Fas resistance, due
either to Fas mutations or secretion of decoy receptors,
coupled with the ability of tumor cells to express Fas
ligand effectively enables a preemptive strike or “counterattack” against the host immune system (129).
The recent finding that tumor cells are more sensitive to TRAIL-induced apoptosis than normal cells generated considerable interest (131). Moreover, the discovery of novel truncated TRAIL receptors in normal
tissues, but not in most cancer cells provided the first
evidence that cells can protect themselves against
death receptor-triggered death, a finding that could be
exploited for the specific killing of tumor cells (131). In
particular, TRAIL-induced killing could perhaps provide the long sought-after means to trigger p53-
independent apoptosis in tumor cells, which lack wildtype p53. Another finding, which sparked hopes of
selective targeting of cancer cells, was the identification of a novel IAP homologue, designated survivin
(132). Survivin was undetectable in terminally differentiated adult tissues, but abundantly expressed in
transformed cell lines and in all common human cancers, suggesting that apoptosis inhibition may be a
general feature of neoplasia. Similarly, the expression
of several IAPs in human malignant glioma cell lines
was suggested to play an important role in the resistance of these cells to apoptotic stimuli that directly
target caspases (133). These findings may explain why
cancer cells are impervious to signals that trigger
apoptosis, and also offer an ideal target for apoptosisbased cancer therapy: if survivin or related IAPs are
inactivated through therapeutic intervention then perhaps cancer cells can be made more susceptible to
conventional cancer therapy. Finally, inhibition of the
transcription factor NF-kB through adenoviral delivery of IkBa was recently shown to sensitize chemoresistant tumors to treatment resulting in tumor regression (134). These data suggest that inhibition of NF-kB
and its associated anti-apoptotic activity, which may
depend, in part, on the transcriptional activation of the
survivin-related molecules cIAP1 and cIAP2, could be
useful as an adjuvant therapy in cancer treatment.
Angiogenesis. Angiogenesis is the process leading
to the formation of new blood vessels, and it has been
proposed that tumor growth is critically dependent on
such neovascularization. Hence, angiogenesis inhibitors are known to impair metastatic growth and sustain dormancy of tumors by indirectly increasing apoptosis (135). Cyclooxygenase-2 (COX-2), a key enzyme in
regulation of eicosanoids, was shown to be inducible by
a variety of apoptotic stimuli and to serve as an inducer
of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), two regulators of angiogenesis (136). COX-2 also induces nitric oxide synthase
(iNOS), which in turn is involved in the regulation of
vascular permeability. Suppression of COX-2 activity
and reconstitution of iNOS expression render tumor
cells sensitive to chemotherapy treatment (137). Moreover, the combination of radiation therapy with angiostatin, a proteolytic fragment of plasminogen that
inhibits angiogenesis, was shown to target tumor vasculature in vivo without an increase in toxicity toward
normal tissues (138). Benjamin et al. (139) have recently demonstrated the selective vulnerability and
death by apoptosis of immature tumor vessels following withdrawal of vascular endothelial growth factor
(VEGF). Collectively, these findings indicate that neovascularization is the Achille’s heel of tumors, and
suggest that angiogenesis-based therapy might be an
attainable goal also in the treatment of human cancer.
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ATHEROSCLEROSIS
Atherosclerosis is characterized by thickening of the
arterial intima leading ultimately to cardiac and cerebral infarction (discussed below). Several cell types
present in atherosclerotic lesions, including endothelial cells, smooth muscle cells and macrophages, are
known to undergo apoptosis with varying outcome. For
instance, apoptotic death of endothelial cells has been
suggested to be an initial step in the development of
atherosclerosis (for review see 140). This hypothesis is
supported by the observation that pro-atherosclerotic
factors such as angiotensin II, oxidized low density
lipoprotein, reactive oxygen species and inflammatory
cytokines all induce apoptosis in endothelial cell,
whereas known protective factors such as estrogen and
nitric oxide prevent apoptosis in these cells. Furthermore, apoptosis is involved in the turnover of lipidladen foam cells of both macrophage and smooth muscle cell lineage in atherosclerotic lesions in murine
models of atherosclerosis (141). Cai et al. (142) have
reported evidence that Fas may regulate apoptosis of
foam cells in advanced human atherosclerotic lesions.
Animal studies show that activation of the nitric oxide
synthase induces apoptosis of vascular macrophages in
intimal lesions, and this was suggested to play a role in
the regression of atherosclerosis (143).
Loss of smooth muscle cells in vulnerable regions of
an atherosclerotic plaque may jeopardize the integrity
and stability of the plaque. Importantly, apoptosis of
vascular smooth muscle cells has been demonstrated in
atherosclerotic plaques (144). Furthermore, it is likely
that apoptotic vascular smooth muscle cells can promote thrombosis directly by exposure of phosphatidylserine on the cell surface (144). Mallat et al. (145)
observed high levels of shed membrane microparticles
(apoptotic bodies) with procoagulant potential in human atherosclerotic plaques, suggesting a role for
apoptosis in plaque thrombogenicity. We found that
human aortic smooth muscle cells treated with 25hydroxycholesterol, an oxidation product of cholesterol,
undergo apoptosis with activation of caspase-3 (146).
Apoptosis was potentiated by TNFa and IFNg, suggesting that the effects of lipid oxidation products on
cells in atherosclerotic lesions may be regulated by
inflammatory cytokines present in the microenvironment. Furthermore, apoptosis was inhibited by the
calcium channel blockers verapamil and nifedipine,
thus serving to underscore the role of calcium as a
second messenger in apoptosis signaling.
Accelerated atherosclerosis of the coronary arteries
following chronic rejection is the most common cause of
heart transplant failure. Recent studies have indicated
a role for anti-apoptotic genes, including Bcl-X L, in the
prevention of chronic cardiac graft rejection (147). In
addition, induction of heme oxygenase-1 (HO-1), the
rate-limiting enzyme in the degradation of heme, also
protected allografts against chronic injury and transplant atherosclerosis. These data concur with previous
findings in mice with a targeted disruption of HO-1, in
which prolonged survival of cardiac xenografts could
not be achieved (148). More recently, HO-1 was shown
to afford protection against apoptosis by virtue of its
augmentation of iron efflux from cells (149). These
findings may allow for the development of new strategies to control graft rejection.
STROKE AND CARDIAC DISEASE
Stroke. Neuronal death in stroke has previously
been attributed to necrosis. However, numerous studies have indicated that apoptosis may also contribute
to neuronal demise in oxygen-deprived brains (44).
Moreover, several studies conducted in animal models
have implicated caspases in neuronal cell death after
stroke. Hara et al. (150) showed that intraventricular
injection of caspase-1 inhibitors can reduce ischemic
neuronal loss (although, considering the role of
caspase-1 in cytokine processing and inflammation, the
reduction in cell death evidenced in these studies may
perhaps reflect an inhibition of inflammatory rather
than apoptotic damage). More recently, systemic or
intraventricular injection of a pan-caspase inhibitor or
a caspase-3-selective caspase inhibitor afforded neuroprotection in animal models of neonatal hypoxicischemia (151) and focal ischemic infarction (152).
Caspase-3 activity was also shown to increase after
experimental cerebral ischemia in rodents (153) and
XIAP overexpression prevented the activation of
caspase-3 under these conditions, thus allowing neurons to survive after the ischemic insult (154). Furthermore, a breach in the mitochondrial barrier and the
release of mitochondrial caspase-9 was demonstrated
in an animal model of cerebral ischemia, suggesting
that mitochondrial events may be central in postischemic neuronal damage (155). Suppression of apoptosis may prove particularly useful in acute disease
processes such as stroke and we anticipate a rapid
translation of anti-apoptotic therapies from the laboratory to the clinic. Indeed, drugs that inhibit apoptosis
may be effective in the prevention of reperfusioninduced injury (discussed below) even when administered some time after the stroke or myocardial infarction has occurred.
Ischemia/reperfusion. Ischemia, i.e., oxygen starvation, of tissues during arterial occlusion, shock and
organ transplantation is a common and important
cause of death (for review see 156). Following the initial insult, cells may die rapidly by necrosis as a result
of hypoxia and decline of energy levels. Additional loss
of parenchymal cells by apoptosis may occur during
reperfusion of the affected organ (156). Fas/Fas liganddependent mechanisms have been proposed to account
at least for some forms of reoxygenation-induced apop-
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tosis. Rasper et al. (157) reported that the caspase
inhibitor usurpin (also known as FLIP) was abundantly expressed in cardiac and skeletal muscle and
was downregulated in cardiac tissue following
ischemia/reperfusion injury in vivo. The distribution of
usurpin was the reciprocal of that of active caspase-3,
which was abundant in apoptotic, infarcted tissue and
low in unaffected regions of the heart. Usurpin may
therefore act as a modulator of apoptosis sensitivity in
cardiac myocytes following ischemia/reperfusion injury. ARC (apoptosis repressor with caspase recruitment domain) is another apoptosis-regulating molecule expressed exclusively in skeletal and cardiac
muscle (158). ARC was shown to selectively target
caspases, and delivery of ARC by gene transfer or
enhancement of its endogenous activity may serve as a
novel strategy for treatment of diseases with excessive
muscle cell apoptosis.
Heart failure. Evidence for a role of caspases in
cardiac development comes from recent knockout studies in mice (for review see 47). Deletion of the mouse
caspase-8 gene resulted in impaired heart muscle development and death in utero, presumably from cardiac failure. Similarly, gene targeting of the caspase
adaptor protein, FADD, resulted in a lethal phenotype
with profound signs of cardiac failure and hemorrhage.
Apoptosis may also play a role in cardiac disease such
as congestive heart failure, a major cause of cardiovascular hospitalization. Congestive heart failure is defined as the inability of the heart to provide adequate
blood flow, oxygen, and nutrients to tissues and organs
(159). Regardless of the origin of the cardiac insult
leading to congestive heart failure, such as myocardial
infarction, ischemic cardiomyopathy and hypertension,
the progression of heart failure is invariably manifested as cardiac remodeling with loss of left ventricular mass. The mechanisms responsible for the thinning
of ventricular tissue are poorly understood, but may, at
least in part, occur by apoptosis of cardiac myocytes.
For instance, evidence was recorded for apoptotic myocyte loss in cardiomyopathy associated with certain
arrythmias and in ischemic and dilated cardiomyopathies of end-stage heart failure (159). Moreover, apoptosis of myocytes in patients with intractable congestive heart failure was demonstrated despite the enhanced expression of Bcl-2 in these cells (160). Cardiac
myocytes and their skeletal muscle counterparts are
characterized by an unusually high density of mitochondria, and we recently showed that skeletal muscle
cells apparently do not express APAF-1, the protein
“platform” required for post-mitochondrial caspase activation (161). By contrast, Narula et al. (162) provided
evidence for mitochondrial cytochrome c release and
activation of caspase-3 in end-stage human cardiomyopathy, suggesting a molecular basis for novel inter-
ventional strategies in conditions with myocardial dysfunction.
TRANSPLANTATION
Graft rejection is a potential threat in all cases of
tissue transplantation. However, there are certain immune privileged sites in the body, such as the testis
and the anterior chamber of the eye, where allogeneic
or xenogeneic tissue grafts enjoy prolonged survival
relative to other areas. While immune privilege was
originally perceived as a passive process relying on
physical barriers and isolation, recent data support the
view of immune privilege as an active phenomenon.
Specifically, a number of recent papers have indicated
a role for Fas in maintaining the immune privileged
state and implicated the Fas system in both the regulation of physiological cell turnover and the protection
of particular tissues against lymphocyte-mediated
damage (163). Thus, testicular allografts expressing
functional Fas ligand evaded rejection, presumably by
inducing apoptotic cell death of Fas expressing recipient T cells activated in response to graft antigens (164).
Similarly, interactions between Fas and its ligand are
important for the maintenance of immune privilege in
the eye (165). A recent report described the prevention
of rejection of insulin-producing islet cells in diabetic
mice by cotransplantation of syngeneic myoblasts genetically engineered to express Fas ligand (166). While
some of these findings have been disputed by other
investigators (167) they nevertheless provide a rational for the manipulation of the local environment of a
graft in a highly site- and immune-specific manner,
and could represent the beginning of a new era in
transplantation immunology. For instance, it may be
feasible to endow allografts or xenografts with the
property of immune privilege, or resistance to rejection, by expressing Fas ligand (and possibly other
death ligands such as TRAIL) in the donor tissues, as
outlined above. Indeed, the development of a “universal” graft which any recipient could accept, such that
the tissue would not be destroyed in a transplant rejection, would be paramount to attaining “the Holy
Grail of transplant biology” (168).
AGING
The process of aging should be distinguished from
the diseases of aging such as cancer: the phenotype of
aging affects all individuals in a population while diseases of aging affect only a subset (169). Several studies have shown that single gene mutations may significantly extend the life-span of fruit flies and nematodes
(for review see 169). For instance, age-1 mutants of C.
elegans have increased levels of SOD and catalase, are
more resistant to oxidative stress and live twice as long
as wild-type nematodes. Similarly, the long-lived Dro-
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sophila mutant methuselah is resistant to oxidative
stress as well as starvation. In humans, on the other
hand, heritability of life-span is relatively minor (170).
Nevertheless, several alternative molecular models of
aging exist which may be applied to the human species.
The “free radical” theory of aging, for instance, states
that reactive oxygen species, which are generated by
cellular metabolism, cause cumulative damage over a
lifetime. Genome instability, i.e., the accumulation of
genomic changes over time, has also been suggested as
a cause of aging. Much attention has focused on the
reactivation of the telomerase enzyme in cultured human cells, which can extend the replicative life-span of
these cells beyond the normal limit (171). However, it
remains to be tested whether telomere shortening and
cellular senescence are causally related to aging. Finally, dysregulated apoptosis may contribute to aging,
although genetic studies in C. elegans appear to argue
against a relationship between programmed cell death
and senescence, at least in nematodes (172). Mitochondria are of particular interest in this context, given the
evidence for the role of these organelles in apoptosis
(173). Mitochondrial function apparently deteriorates
during normal aging, as evidenced by a decline in electron transport, accumulation of mitochondrial mutations, and decreased bioenergetic capacity (measured
by mitochondrial membrane potential) (174). Hence,
an accumulation of mitochondrial DNA mutations has
been observed in aging as well as in cancer and is due,
most likely, to oxidative damage, which increases with
age. Cells bearing pathogenic mitochondrial mutations
are sensitized to oxidative stress, which in turn is
known to induce permeability transition, a putative
central regulator of apoptosis (174).
Down’s syndrome serves as an example of premature
or pathological aging (175). These individuals have a
decreased life expectancy and display many progeroid
features, including neurohistopathological changes
similar to those found in Alzheimer’s disease (i.e., senile plaques and neurofibrillary tangles). An in vitro
study on Down’s syndrome patient cells provided evidence for neuronal apoptosis, which was caused by a
defect in the metabolism of reactive oxygen species
(176). To conclude, (systemic) aging may perhaps be
viewed as a mitochondrial disease associated with oxidative stress and dysregulated (cellular) apoptosis. On
the other hand, the inability of cells to undergo apoptosis may also be related to aging and more specifically
to the diseases of aging, since senescent, precancerous
cells may accumulate under such conditions. By this
interpretation, “successful aging” might depend on the
maintenance of apoptotic mechanisms to eliminate
cells that have sustained DNA damage (175). Further
studies are needed before the role of apoptosis in aging
is understood.
APOPTOSIS AS A THERAPEUTIC TARGET
Does apoptosis occur in human disease? While evidence for the involvement of dysregulation of apoptosis
appears readily forthcoming in some cases (for instance, defective apoptosis in ALPS due to inherited
Fas mutations) in other cases, such evidence is merely
circumstantial, and often based on crude methods of
apoptosis detection in postmortem clinical specimens.
An illustrative example is provided by recent observations in Creutzfeld-Jacob’s disease, a neurodegenerative condition characterized by the accumulation of a
post-transcriptionally modified pathological form of
the host-encoded prion protein. A prion protein fragment was shown to induce apoptotic cell death in vitro
in primary rat hippocampal neurons (177) and the
normal cellular isoform of the prion protein serves to
protect against apoptosis in vivo in an animal model of
prion disease (178). However, Ferrer (179), in a study
of human brain specimens from deceased patients using in situ end labeling of fragmented DNA, has shown
that none of the positive cells displayed apoptotic morphology. Moreover, deposits of prion protein were not
associated with increased staining of fragmented DNA.
These findings suggest that the mere presence of DNA
fragmentation in clinical specimens does not necessarily indicate apoptosis, a conclusion supported by other
investigators in the field (180). Indeed, while caspase
activation may be viewed as an integral feature of
apoptotic cell death (181), even the detection of activated caspases or cleavage of typical caspase substrates may not suffice to label cells as “apoptotic.” For
example, caspase activation in heart muscle cells may
occur despite the conspicuous absence of other indices
of apoptosis (162). In addition, caspase activation and
cleavage of the classical caspase substrate, poly(ADPribose) polymerase, has been demonstrated in activated, non-apoptotic T lymphocytes (182). Therefore,
great care should be taken to assess apoptosis by both
morphological and biochemical criteria and attempts
should also be made to devise improved methods for
the detection of apoptosis in vivo and in fixed pathological specimens.
Does apoptosis play a role in disease pathogenesis?
The view that disease can result from the dysregulation of active cellular suicide mechanisms represents a
novel paradigm in medicine. A critical question, therefore, is whether the labeling of dead cells as “apoptotic”
has added to our understanding of disease processes, or
whether this is merely, in the words of the Canadian
singer/songwriter Leonard Cohen, a new skin for the
old ceremony? Indeed, to establish that apoptosis occurs (or is lacking) in a particular disease does not
mean to say that apoptosis defects are directly implicated in the pathogenesis of the condition and therefore a legitimate target for therapeutic intervention,
i.e., involvement of apoptosis cannot a priori be
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FIG. 2. Coup de grace: Therapeutic strategies based on modulation of apoptosis.
equated with causality (183). For instance, it could be
argued that the primary cause of T cell depletion in
AIDS is not apoptosis per se but rather the inability of
lymphopoiesis to replace the CD4 1 T cells lost prematurely because of the infection with HIV. In this case,
the enhanced rate of T cell apoptosis in cells obtained
from HIV-infected persons may merely be reflective of
a chronic immune activation in these individuals,
rather than being involved in the pathogenesis of the
disease itself. Similarly, the recent finding that cells
from patients with Niemann–Pick’s disease are resistant to radiation-induced apoptosis due to an inherited
deficiency in acid sphingomyelinase activity (184) does
not necessarily imply that these aberrations are associated with the development or progression of disease.
Therefore, the present challenge lies in the identification of those conditions in which apoptosis plays a
causal role, that is, those diseases in which cellular
self-annihilation is linked to the pathogenesis or progression of disease and not merely a consequence of
underlying mechanisms.
Can apoptosis be safely targeted for therapy? An
important question is how to harness apoptotic processes for therapeutic benefit while maintaining an
adequate degree of apoptosis in unaffected “bystander”
tissues. Indiscriminate inhibition of apoptosis could
lead to the survival of genetically damaged cells which
would otherwise die, thus causing widespread hyperplasia, whereas inappropriate promotion of apoptosis
might lead to undesirable tissue degeneration (185).
Indeed, Wickremasinghe and Hoffbrand (186) caution
that anti-cancer strategies dependent solely on the induction of apoptosis may result in the rapid evolution
of clones resistant to killing. Nevertheless, one may
envisage a number of principle categories of apoptosisregulating therapies (185, Fig. 2), which may be utilized alone or in conjunction with conventional treatments: (a) injectable molecules targeted at upstream
modulators of apoptosis, such as death receptor decoys
or soluble death ligands; (b) small molecule pharmaceuticals designed to regulate expression or activation
of apoptosis-related genes and gene products, including Bcl-2, caspase family members and p53; and (c)
gene therapy, e.g., overexpression of pro-apoptotic
Bcl-2 family members or replacement of p53. The first
clinical results of p53 reconstitution by gene therapy
were reported by Roth et al. (187), who obtained tumor
regression in three patients and tumor stabilization in
three others out of nine non-small cell lung cancerafflicted individuals with p53 mutations. Utilizing adenoviral vectors as an alternative delivery system,
Bischoff et al. (188) were able to induce complete regression in 60% of implanted tumors in nude mice
bearing human cervical carcinoma tumors. These authors used an E1B 55 kDa-deficient adenovirus which
is only able to replicate in and kill p53-deficient cells,
and these results therefore demonstrate the feasibility
of using anti-tumor agents that kill only p53-deficient
tumor cells, leaving normal cells unaffected. Future
work should aim at the design of therapies specifically
targeted against components of the apoptosis pathway
in diseased tissues while leaving bystander tissues unscathed.
Does prevention of apoptosis restore function? Another important question to consider is whether cells
rescued from apoptosis are functional or whether they
are simply “undead” or anergic and, as a result of
inadequate expression of recognition signals, not
readily available for phagocytic clearance. In other
words, rescuing a cell from death may not necessarily
be the same thing as preserving its function. Indeed, it
was recently shown that in cells, which received signals via the Fas receptor in the presence of caspase
inhibitors, the phenotype of death was merely switched
from apoptosis to necrosis (189). Similarly, in cells
overexpressing Bcl-2, cell death induced by oxidized
low density lipoproteins shifted from apoptosis to necrosis (190). It is also conceivable that apoptosis is so
far down the cascade of irreversible and irreparable
changes in some diseases that blocking this process
may not halt the progression of the disease itself. In
fact, interference with the orderly phagocytic clear-
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ance, which is part of the apoptotic program, could lead
to necrotic cell lysis, which would trigger harmful inflammation. The recent report of Fas-dependent apoptosis and subsequent pulmonary fibrosis in mice (191)
may perhaps be interpreted as a case of excessive
apoptosis which has overwhelmed the phagocytic capacity of the tissue with ensuing “secondary” necrosis
and inflammation. However, an elegant study in the
fruit fly indicates that suppression of apoptosis may, in
fact, constitute a viable therapeutic approach. Davidson and Steller (192) employed a Drosophila model of
retinitis pigmentosa, and showed that the retinal degeneration due to mutations in either rhodopsin or a
rhodopsin phosphatase gene occurred by apoptosis and
could be blocked by caspase inhibitors with subsequent
restoration of visual function in otherwise blind flies.
Further proof of the principle that the suppression of
apoptosis can result in improved cellular function was
provided by Jilka et al. (193), who demonstrated that
the increased bone mass caused by administration of
parathyroid hormone (PTH) is due, at least in part, to
the salvage of osteoblasts by prevention of apoptosis.
To conclude, we are confident that in terms of understanding the pathogenic mechanisms underlying human maladies, we are not at the fin de siècle. We are,
instead, at a new beginning. Moreover, we anticipate
that apoptosis-based strategies will be rapidly incorporated into our future therapeutic arsenal.
ACKNOWLEDGMENTS
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
This study was supported by the Swedish Medical Research Council (S.O.) and the Swedish Cancer Foundation (B.Z.). B.F. currently
holds a combined clinical training and research position at the Karolinska Hospital and Karolinska Institutet.
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