Apoptosis: Mechanisms and Clinical Implications: Reviewarticle

Anaesthesia, 2000, 55, pages 10811093
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R E V I E W A RT I C L E
Apoptosis: mechanisms and clinical implications

P. C. A. Kam1 and N. I. Ferch2
1 Clinical Associate Professor and Senior Staff Anaesthetist, Department of Anaesthesia and Pain Management,
University of Sydney, Royal North Shore Hospital, St Leonards, NSW 2065, Australia
2 Anaesthetic Registrar, Department of Anaesthetics, University of Sydney, Royal Prince Alfred Hospital, Camperdown,
NSW 2050, Australia
Summary
The balance between cell survival and death is under tight genetic control. A multiplicity of
extracellular signals and intracellular mediators is involved in maintaining this balance. When the cell
is exposed to physical, biochemical or biological injury, or deprived of necessary substances, it
activates a series of stress-response genes. With minimal insults, the cell may recover. With greater
insults, single cell death, or apoptosis, results; the cell dies and is recycled to its neighbours. If the
insult overwhelms a large number of cells then necrosis ensues, with an accompanying inflammatory
response. Dysregulation of the controlling mechanisms of this system results in disease. Deficient
apoptosis is associated with cancer, auto-immunity and viral infections. Excessive apoptosis is
associated with ischaemic heart disease, stroke, neurodegenerative disease, sepsis and multiple organ
dysfunction syndrome. There are myriad therapeutic options unfolding as understanding is gained of
apoptosis and its control.
Keywords Apoptosis. Cell death: morphology; mechanisms; clinical implications.
Multiple organ dysfunction syndrome.
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Correspondence to: Dr P. C. A. Kam
Accepted: 4 April 2000
Cell death, a tightly controlled, finely orchestrated event,

may be described either as apoptosis or nonapoptotic cell
death, traditionally called `necrosis' [1]. Apoptosis is a
process of cell suicide, the mechanisms of which are
encoded in the chromosomes of all nucleated cells.
Physiological cell death that removes unwanted cells plays
an important role in development, tissue homeostasis and
defence against viral infection and mutation. Apoptosis is
regulated by complex molecular signalling systems. Tissue
ischaemia and reperfusion activate these molecular
systems, which therefore represent a therapeutic target
for novel treatment to preserve cellular integrity in critical
organs such as the brain and heart. Apoptotic cells
undergo orderly, energy-dependent enzymatic breakdown into characteristic molecular fragments, deoxyribonucleic acid (DNA), lipids and other macromolecules,
which are packaged into small vesicles that may be
phagocytosed and reused. The cells involute and die with
minimal harm to nearby cells [2]. In contrast `necrotic'
q 2000 Blackwell Science Ltd
cell death is characterised by inflammation and widespread damage.

Apoptosis has a central role in the pathogenesis of
human disease when the genes controlling the apoptotic
process are suppressed, overexpressed or altered by
mutation [3]. Disordered apoptosis is implicated in a
variety of human diseases (Table 1). Research into
apoptosis is proceeding at a fast pace and this has led to
the possibility of new therapeutic approaches to some
intractable human diseases [4].
The purpose of this review is to provide a background to
the molecular components that activate apoptosis and the
relevance of these to a variety of human diseases, and to
discuss the potential for novel therapies based on our
understanding of them. An understanding of the role of cell
injury and death in the pathophysiology of organ dysfunction is relevant to anaesthetists and intensivists particularly in
the management of ischaemic and reperfusion injury, and
multiple organ dysfunction syndrome (MODS).
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P. C. A. Kam and N. I. Ferch Apoptosis

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Table 1 Human diseases associated with

Increased apoptosis
Central nervous system
Myocardium
Lymphocytes
Macrophages
Decreased apoptosis
Epithelial tissues
Blood vessels
Lymphocytes
Haemopoietic system
Degenerative diseases (Alzheimer's and Parkinson's disease)

Cerebral ischaemia
Peri-infarct border zones
Lymphocyte depletion in sepsis and HIV infection
Bacillary dysentery (Shigella dysenteriae)
disordered apoptosis.
Carcinogenesis
Intimal hyperplasia
Autoimmune disorders
Leukaemia, lymphoma
History
In the 1880s Weigert & Cohnheim described the

microscopic appearance of cell death in necrotic tissue
as coagulation necrosis [5]. In 1885, Flemming described
the process of `chromatolysis' in which the nuclei of
mammalian ovarian follicles broke up and ultimately
disappeared in spontaneous cell death [68].
In the early 1970s, Kerr described the electron
microscopic appearance of singe-cell death in the livers
of animals [9, 10] treated with toxins (heliotrine and
albitocin) and ischaemia (by ligation of a large branch of
the portal vein) and called it `shrinkage necrosis' [11].
`Single cell death' in carcinogen-treated rat adrenal cortex
[12] and in tumour cells treated with actinomycin D,
mitomycin C, cytosine arabinose and cycloheximide [13]
was also observed. In 1971, Wyllie found that single cell
death in the adrenal gland was induced by hypophysectomy, and suggested a common pathway for cell death
initiated by hormone regulation and carcinogen-induced
injury [14, 15]. In a landmark paper in 1972, Kerr et al.
described the characteristic sequential changes occurring
in cell structure during the death process in healthy
tissues, normal development, tumour regression, atrophy
and involution [1]. The term àpoptosis' (derived from the
Greek word for `falling off ', a reference to the falling of
leaves from trees in autumn in response to the impending
threat of freezing and damage in winter) was coined [1].
Further momentum was gained in the mid-1980s
when Horvitz reported specific cell death during the
development of the nematode Caenorhabditis elegans and

cloned the C. elegans death genes (ced genes) responsible
[16]. The nematode anti-apoptotic gene ced-9 shared
homology with the mammalian proto-oncogene, Bcl-2
[17], and was shown to preserve B lymphocytes [18, 19].
During the 1990s, the molecular mechanisms that keep
apoptosis in check and the molecular events involved in
disordered apoptosis were unravelled. Rodent fibroblast
tumours expressing human c-myc genes had high rates of
apoptosis [20], suggesting that cancer can occur when a
mutation results in dysfunctional apoptosis [21].
Definitions
Apoptosis specifically refers to an energy-dependent,

asynchronous, genetically controlled process by which
unnecessary or damaged single cells self-destruct when the
apoptosis genes are activated [22, 23]. Briefly, the cell
shrinks and detaches from neighbouring cells and the
nucleus is broken down. The nuclear fragments and
organelles condense and are ultimately packaged in
membrane-bound vesicles, exocytosed and ingested by
surrounding cells. Membrane integrity is preserved and
dyes are only taken up late in the process. The absence of
inflammation differentiates apoptosis from necrosis. The
differences between apoptotic and necrotic cell deaths are
summarised in Table 2.
Ischaemic and accidental (caused by toxins) cell death
are characterised by cell swelling or òncosis' (from the
Greek word ònkos' meaning swelling), resulting in
Table 2 Differences between apoptosis and
Apoptosis
Necrosis
Physiological or pathological
Asynchronous process in single cells
Genetically controlled
Late loss of membrane integrity
Cell shrinkage
Condensation of nuclear contents
(`ladder' formation of chromatin)
No inflammatory reaction
Always pathological
Occurs synchronously in multiple cells
Caused by overwhelming noxious stimuli
Early loss of membrane integrity
Generalised cell and nucleus swelling
Nuclear chromatin disintegration
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necrosis.
Inflammatory reaction

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cytoplasmic and nuclear swelling and karyolysis (loss of

affinity for basic dyes). After either oncosis or apoptosis,
cells reach the stage of necrosis where phagocytosis
occurs, and in the case of oncosis, this is accompanied by
inflammation. Apoptosis and oncosis/necrosis are part of
an overlapping spectrum [5, 8, 2426]. Cells exposed to
an overwhelming noxious stimulus, such as ischaemia or
toxins, undergo oncosis and necrosis [27]. Programmed
cell death (PCD) refers to cell suicide occurring during
normal embryological development of an immature
organism and the maturation of tissues or organs [28,
29] and does not require de novo gene expression [30].
Pathophysiology
Morphological features
There are three distinct phases of apoptosis [31]. During
the first phase, the cell detaches from its substratum and
adjacent cells with a loss of microvilli and junctional
complexes or desmosomes [32]. The DNA is digested by
specific endonucleases into fragments and ultimately
packed into vesicles. The changes in DNA include strand
breakage (karyorhexis) and condensation of nuclear
chromatin (pyknosis). This pyknotic chromatin appears
as characteristic crescent-shaped `caps' under light
microscopy. The endoplasmic reticulum swells and
exocytoses its contents. The cell becomes denser as the
cytoplasm shrinks and involutes. In the second phase, the
cell produces pseudopodia (budding) which contain
organelles or nuclear fragments, and these break off into
multiple membrane-bound vesicles. The remaining cell
becomes a round, smooth membrane-bound remnant
(apoptotic body) [3, 4, 32]. In the third phase, the cell
membrane becomes permeable to dyes such as Tryphan
Blue. The apoptotic body and membrane-bound buds
may then be phagocytosed by macrophages, epithelial
cells, vascular endothelium or tumour cells. The entire
process occurs may take only 15 min, and therefore may
be undetectable on tissue sections [5, 23, 33].
In contrast, oncosis is characterised by cellular and
organelle swelling with late nuclear fragmentation and
breakdown by lysosomal enzymes. The swelling is caused
by a deficit in adenosine triphosphate (ATP) production

that leads to membrane ionic pump failure and increased
plasma membrane permeability. This results in bleb
(blister-like, fluid-filled structures) formation of the
plasma membrane and ultimate rupture [5]. There is an
influx of neutrophils and macrophages in the surrounding
tissues, leading to generalised inflammation.
Techniques to identify and quantify apoptosis include
staining with nuclear stains such as Hoechst 33258 that
allow visualisation of nuclear chromatin clumping.
Modern video microscopy allows visualisation of the
temporal sequence of events that occur over 1560 min
to 24 h [23, 34, 35].
Fluorescent microscopy using acridine orange allows
characteristic chromatin clumping to be observed soon
after staining. A further refinement is the comet assay,
which shows up DNA degradation [2]. More accurate
identification of apoptosis is achieved with methods that
specifically target the characteristic DNA cleavages [36].
Agarose gel electrophoresis of extracted DNA fragments
yields a characteristic `ladder' pattern which can be used
as a marker for apoptosis. A lesser extent of DNA
degradation produces hexameric structures called
`rosettes' [4]. Necrotic cells leave a nondescript smear
[3]. The terminal transferase deoxyuridine nick-end
labelling of DNA breaking points (TUNEL) method
[37], which labels the uridine residues of the nuclear
DNA fragments, can be applied to tissue sections and
cell cultures to quantify apoptosis. Flow cytometry assays
[3840] may prove to be most accurate in quantifying
apoptosis.
Mechanism of cell death
Following an appropriate stimulus, the first stage or
`decision phase' of apoptosis is the genetic control point
of cell death. This is followed by the second stage or
èxecution' phase, which is responsible for the morphological changes of apoptosis.
There are four main groups of stimuli for apoptosis
[4, 30, 32, 34, 4145]. A wide variety of physiological
and pathological stimuli can initiate apoptosis and these
stimuli are summarised in Table 3. The first group of
Table 3 Stimuli for apoptosis.

DNA (genome) damage
Activation of death receptors
Stimulation of apoptotic pathway
Direct physical cell damage
Ionising radiation
Anti-cancer drugs (e.g. alkylating agents)
Binding of `death receptors' (e.g. Fas receptor, TNF receptor)
Withdrawal of growth factors (e.g. nerve growth factor, IL-3)
Phosphatases, kinase inhibitors
Heat, ultraviolet light, oxygen free radicals, hydrogen peroxide
DNA, deoxyribonucleic acid; TNF, tumour necrosis factor; IL-3, interleukin-3.
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stimuli causes DNA damage and include ionising

radiation and alkylating anticancer drugs. The second
group induces apoptosis via receptor mechanisms, either
by receptor activation mediated by glucocorticoids (acting
on the thymus) [34], tumour necrosis factor-a (TNF-a),
or by withdrawal of growth factors (nerve growth factor
and interleukin (IL)-3) [41, 42]. The third group
comprises biochemical agents that enhance the downstream components of the apoptotic pathway and includes
phosphatases [30] and kinase inhibitors (e.g. calphostin C,
staurosporine). The fourth group comprises agents that
cause direct cell membrane damage and includes heat,
ultraviolet light and oxidising agents (superoxide anion,
hydrogen peroxide). Excessive production of reactive
oxygen species (ROS), such as superoxide, hydrogen
peroxide and the hydroxyl radicals, produces free radicals
that damage lipid membranes, proteins, nucleic acids and
extracellular matrix glycosaminoglycans. Many of these
stimuli cause necrosis in larger doses. Injury to cell
membranes induces apoptosis by activating acid sphingomyelinase that generates the second messenger ceramide
from membrane lipids [43, 44].
The signal that initiates apoptosis may result from
binding of a cell-surface `death' receptor or from damage
to the genome. Death receptors that initiate apoptosis
include the Fas receptor and the TNF receptor system
[41]. The Fas receptor, initially known as CD95 or
APO-1, is a transmembrane glycoprotein death receptor
that is activated by binding of Fas ligand (Fas-L) to cell
membranes [41]. Intracellular molecules known as Fasassociated death domain (FADD) are produced. Fas
receptors are found in epithelial tissues, tumours and
haemopoietic tissues, and may be induced in other tissues
that do not express them. The Fas pathway is important in
controlling the immune response. Cytotoxic T lymphocytes expressing Fas ligands activate cells bearing Fas
receptors and induce apoptosis.
The TNF receptor system mediates different biochemical pathways [41, 42]. A TNF-related apoptosis-inducing
ligand (TRAIL) has been discovered. Cancer cells are
susceptible to TRAIL-induced apoptosis. Following
binding of the TNF receptor, intracellular molecules
called `death domains' are produced. A TNF receptor
associated death domain (TRADD) has been identified.
Tumour necrosis factor may suppress apoptosis by binding
to the receptor, TNFR2, which activates a protein known
as Nuclear Factor kB (NF-kB), classed as an inhibitor of
apoptosis protein (IAP) that prevents the execution phase
of apoptosis [43]. It is a DNA binding protein that
regulates many pro-inflammatory genes for the production of cytokines and other pro-inflammatory molecules.
There is increasing evidence that NF-kB is important in
the pathogenesis of systemic inflammatory response
1084
syndrome (SIRS), MODS and acute respiratory distress

syndrome (ARDS) [28].
Decision phase (genetic control)
Apoptosis is controlled genetically and two genes, Bcl-2
and p53 are important. The first, Bcl-2, is a family of genes
that regulates apoptosis [4649]; found on the mitochondrial membrane, endoplasmic reticulum it may
control calcium channels. It is now recognised that
there is a family of mammalian proteins similar to Bcl-2
that promotes or inhibits apoptosis [50, 51]. Proteins such
as Bcl-2 and Bcl-xL prevent apoptosis, whereas Bcl-2
associated x proteins (Bax) such as Bax, Bad, Bak and
Bcl-xS promote apoptosis [30, 52, 53].
The gene p53 is a 53-kDa nuclear phospho-protein
that binds to DNA to act as a transcription factor, and
controls cell proliferation and DNA repair [54]. Mutations
of p53 have been found in . 50% of human cancers (e.g.
colon carcinoma) and are associated with resistance to
treatment [55]. The gene c-myc is a proto-oncogene that
encodes a sequence-specific DNA-binding protein that
acts as a transcription factor and induces apoptosis in the
presence of p53. The c-myc protein is elevated in many
tumours [4].
Mutation of the gene for neuronal apoptosis inhibitory
protein (NAIP) occurs in people with spinal muscular
atrophy [56]. NAIP protects various cells from apoptosis
caused by TNF-a, free radicals and growth factor
deprivation.
Execution phase
The central events in apoptosis are proteolysis and
mitochondrial inactivation. Cellular disruption results
from activation of a family of cysteine proteases called
caspases (CASP) [30, 32, 43, 5759]. Caspases are
proenzymes that have been conserved from nematodes
to humans. Ten human caspases (CASP 110) have been
described. Early molecular studies on apoptosis focused
on the nematode C. elegans and the gene required for the
execution of apoptosis, called ced-3, has been identified
and coded. There are two subfamilies of caspases, the
ced-3 subfamily (produced by ced-3 gene) and the ICE
(IL-1b-converting enzyme) subfamily [60, 61]. Caspase 1,
which is related to ICE, is mainly involved in inflammation [60, 61]. The ced-3 caspases are important effectors
of apoptosis. Caspase 8 or FADD-like interleukin converting enzyme (FLICE) is the most important enzyme of
the ced-3 subfamily [61, 62]. The actions of the caspases
are varied; some are endonucleases that cleave DNA,
some cleave cytoskeletal proteins and others cause a loss of
cell adhesion.
The integrity of the plasma membrane of the apoptotic
cell is maintained initially, although `budding' of the cell

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membrane can occur later. There is no leakage of

lysosomal enzymes that can damage nearby cells or elicit
immune responses [63]. The apoptotic cell expresses
membrane signals that induce phagocytosis [64]. Macrophages can recognise neutrophils undergoing apoptosis
via complexes involving thrombospondin receptors
(CD36) and the avb3 integrin [6567].
Clinical relevance of apoptosis
Central nervous system

In the developing organism there is a specialised form of
cell death previously called apoptosis but now known as
`programmed cell death' (PCD) [30]. During embryonic
development of the nervous system a surplus of cells is
produced. PCD eliminates those neurons whose axons fail
to reach the target. It occurs with the withdrawal of
trophic substances, such as nerve growth factor, or with a
loss of synaptic contact or afferent input. Cytokines (e.g.
TNF-a) and ROS may trigger PCD [68].
Oxidative stress, glutamate excitotoxicity and calcium
influx can induce apoptosis in the mature central nervous
system. Excessive production of ROS causes òxidative
stress', damaging lipid membranes, proteins, nucleic acids
and extracellular matrix glycosaminoglycans. At low
levels of ROS or depletion of antioxidants (e.g. superoxide dismutase, catalase, glutathione peroxidase) apoptosis occurs. At high levels ROS produce more damage
and cause necrosis [30].
Glutamate receptor-mediated neuronal injury is an
important cause of èxcitotoxic` neuronal death following
ischaemia, trauma, epileptic seizures or neurodegeneration [30]. Glutamate produces either necrosis associated
with influx of Na1, Cl2 and water leading to cell swelling
or delayed neuronal death (DND) which appears to be
apoptotic, occurring several hours after exposure and
associated with calcium influx via channels linked to
glutamate receptors [6971].
Calcium is an important second messenger, instrumental in inducing apoptosis by stimulating neurotransmitter release, gene induction and the activation of
enzymes (proteases, phosphatases, protein kinases, endonucleases, phospholipases and nitric oxide synthase) [72].
Phospholipase A2 produces superoxide anion, and nitric
oxide synthase produces nitric oxide, both of which can
cause oxidative stress leading to apoptosis.
Increased apoptosis caused by excessive intracellular
Ca21 has been implicated in cerebral ischaemia, traumatic
brain injury, epilepsy and neurodegenerative disease.
Cerebral ischaemia results in both necrosis and DND.
Cells undergoing DND as a result of less severe insult are
seen on the periphery of the infarct within the penumbra.
There is accumulating evidence that apoptosis plays a role
in DND [30]. Gene transfer using herpes simplex viruses

containing a Bcl-2 vector may offer neuroprotection
against ischaemia [73]. In traumatic brain injury apoptosis
occurs in < 10% of dying neurons, peaking 2448 h after
injury [74]. With severe trauma, the proportion of
necrotic cell death increases and antiapoptotic agents
may minimise cell death following trauma [73].
Enhanced apoptosis is implicated in several neurodegenerative diseases. In Alzheimer's disease accumulation
of b-amyloid peptide in plaques in the brain and cerebral
vasculature causes apoptosis of cortical neurones [75],
possibly via nitric oxide (NO) [76]. Parkinson's disease is
associated with the loss of dopaminergic neurones in the
substantia nigra, and dopamine is thought to cause
apoptosis in exposed neurons [30]. Some forms of familial
amyotrophic lateral sclerosis may be caused by a mutant
gene coding for the antioxidant superoxide dismutase
resulting in motor neuron loss via apoptosis. Future
therapy may involve the use of growth factors or
inhibitors of macromolecular synthesis to block apoptosis
[56].
Defective apoptosis caused by mutant apoptotic genes
may contribute to the development of neural tumours.
Mutated p53 is seen in astrocytic tumours [77, 78].
Protein kinase C inhibitors (e.g. hypericin and calphostin)
can cause apoptosis in glioma cell lines. Gene transfer
techniques have also been used to introduce Bcl-xs into
neuroblastoma cells and ICE retrovirus into gliosarcoma
cells with induction of apoptosis in these tumours
[79, 80].
Cardiovascular system
Recently it has been shown that myocyte death with
cardiac disease occurs by both apoptosis and necrosis in
response to hypoxia and ischaemia [45]. Apoptosis has
been documented in myocardial ischaemia, reperfusion
and infarction, heart failure induced by rapid ventricular
pacing or coronary microemboli, several models of
pressure overload hypertrophy (by passive load on papillary muscle, aortic banding) [42] and ageing [53].
Hypoxia, nutritional deficiency and toxins (e.g.
chemotherapy) that can cause necrosis may induce
apoptosis at lower doses [45, 52]. Infarcted areas have a
central area of necrosis with surrounding apoptosis in the
peri-infarct border zone [42]. Autopsy studies of
myocardial infarction have found apoptotic cells scattered
between the necrotic cells centrally and the normal cells
around the periphery. Coronary artery ligation induces
marked increases in proto-oncogenes (Bcl-2 and Fas) in
myocytes. The initial event during the early hours of
ischaemia appears to be apoptosis. As myocardial cells are
overwhelmed by severe ischaemia, apoptotic genes can no
longer be expressed and necrosis supervenes [81]. In in vivo
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reperfusion studies in rabbit models, apoptosis was seen

predominantly in reperfused hearts, whereas necrosis was
seen mainly with persistent occlusion [82]. Reperfusion
increases free radical production and intracellular calcium,
which are potent inducers of apoptosis.
Apoptosis in vascular smooth muscle cells in atherosclerotic plaques in coronary arteries may be involved in
plaque destabilisation and rupture [83]. Apoptosis is
increased by 43% in primary atherosclerotic lesions and
93% in restenoses [84, 85]. It may be triggered by NO
and is associated with the expression of caspase 1 [84].
Arrhythmias may be associated with deficient apoptosis
during cardiac development. Normally after birth there is
apoptosis of the small round pacemaker cells in the central
and lower part of the AV node. Delay or failure of
apoptosis in these cells may lead to life-threatening
arrhythmias that may resolve spontaneously. Excessive
apoptosis can lead to bradyarrhythmias and sudden death
[45]. Patients with long QT syndrome have increased
rates of apoptosis in the sinus node [85].
A common end-stage pathway of various cardiovascular
diseases results in deteriorating function and cardiac
failure associated with dilated cardiomyopathy [45, 46].
Myocardial hypertrophy may be an initial compensatory
response to overloading of adult myocytes. This is
associated with c-myc, c-fos, and transforming growth
factor, which are also involved in initiating apoptosis.
Dilated cardiomyopathy can be induced by chronic
infusion of TNF-a, which causes apoptosis [43]. A
progressive decline in function in end-stage cardiac failure
may be due to apoptosis and this is supported by the
finding of increased levels of Bcl-2 in the myocytes of
patients with cardiac failure. Although apoptosis itself is
irreversible, minimising it with growth factors and cytokines may prevent the progressive decline in left
ventricular function [45].
Arrhythmogenic right ventricular dysplasia is a cardiomyopathy associated with sudden death. It is postulated
that repetitive episodes of ischaemia and reperfusion
associated with ventricular arrhythmias produce apoptosis, which is found in myocardial biopsies in these
patients [45].
Immune system
Dysfunction of the apoptotic pathway causes autoimmune
disease, immunodeficiencies and lymphoid malignancies.
During development large numbers of precursor cells
from the bone marrow migrate to the thymus. The
majority (9095%) fail to produce T-cell receptor (TCR)
and die via the apoptotic pathway [86]. Thymocyte
apoptosis can be induced by endogenously produced
glucocorticoids and a lack of TCR [87, 88]. Death of a
cell clone (clonal deletion) due to TCR-induced
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apoptosis takes place in immature B cells that produce

antiself antibodies in the fetal liver and later in the bone
marrow [89, 90]. Animal studies indicate that autoimmune diseases, such as rheumatoid arthritis, systemic
lupus erythematosis, inflammatory bowel disease and
insulin-dependent diabetes, may be caused by dysfunctional apoptosis [91, 92]. There is evidence that cytotoxic
T cells kill their target cells by the induction of apoptosis
in the target [93]. Activated peripheral cytotoxic T cells
and peripheral B cells are removed by apoptosis [89].
Granulocyte apoptosis is also important for the
resolution of the inflammatory response. Inflammatory
agents such as lipopolysaccharide (LPS) and granulocyte
colony-stimulating factor (G-CSF) inhibit neutrophil
apoptosis. During apoptosis induced by TNF-a and
NO donors the neutrophil loses its ability to degranulate,
thus limiting the inflammation. Intact senescent neutrophils are engulfed by macrophages and degraded within
minutes, with no release of pro-inflammatory mediators.
When necrotic granulocytes are ingested, the macrophages release mediators leading to inflammation [94].
Haematological diseases, such as myelodysplastic syndromes, aplastic anaemia, chronic neutropenia or severe
b-thalassaemia, are associated with increased apoptosis
within the bone marrow [95].
Viral infection
Many viruses inhibit apoptosis in their target cells to
prolong host cell life and permit viral replication. Viruses
may encode anti-apoptotic proteins, such as the baculovirus IAPs, the baculovirus p35 and cowpox serpin
protein crmA, to promote the development of certain
cancers [96]. DNA viruses also contain anti-apoptotic
genes. For example, the papilloma virus and adenovirus
can encode a p53 inhibitor. Ribonucleic acid (RNA)
viruses may also contain anti-apoptotic genes [97].
Human immunodeficiency virus (HIV) infection is
characterised by a decreased proliferation of T cells with
loss of CD41 cells initially, and loss of CD81 cells, natural
killer cells and neurons later. Inappropriate induction of
apoptosis in HIV-infected CD41 cells is triggered by the
virus [98]. Another viral product, the HIV-1 transactivating protein, Tat, is produced by infected cells and taken
up by uninfected T cells. It enhances apoptosis by
reducing intracellular antioxidant levels, producing oxidative stress [99].
Sepsis and multiple organ dysfunction syndrome
Sepsis is often accompanied by MODS, which may
caused by apoptosis [100]. Systemic release of cytokines
such as TNF-a and IL-1b by bacterial LPS, resulting in
increased intracellular calcium and enhanced production
of oxygen free radicals, is thought to produce apoptosis in

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various organs [101104]. TNF-a may act by causing

macrophage release of IL-1b [105]. In a murine model of
septic shock, direct application of TNF-a caused
apoptosis in hepatocytes [106]. Further support for the
role of apoptosis in endotoxic shock is provided by the
finding that mice deficient in ICE have a marked
resistance to septic shock [107]. It is postulated that
apoptotic (as opposed to necrotic) cell death may confer
advantages to the septic organism by limiting the
inflammatory response [28]. Apoptosis is an important
cause of death of lymphocytes in the immune system
(thymus, lymph nodes and spleen) during bacterial and
viral infections [101, 108113]. Furthermore, apoptotic
cell death has been shown in the lung, ileum, colon and
skeletal muscle during sepsis [109]. Apoptosis may be an
important regulator of a balance between pro- and antiinflammatory factors and this balance is achieved through
widespread lymphocyte apoptosis and subsequently
diminished cytokine load [114].
Trauma patients with and without septic complications
show decreased neutrophil apoptosis associated with
increased tyrosine phosphorylation leading to excessive
tissue damage [115, 116]. Patients with SIRS and
following major elective aortic surgery also show
decreased neutrophil apoptosis, possibly due to circulating
pro-inflammatory anti-apoptotic factors such as LPS,
TNF-a, interferon (IFN)-g, G-CSF and granulocyte/
monocyte colony-stimulating factor [117]. The persistence of neutrophils in such conditions may contribute to
the pathophysiology of MODS through the excessive
release of toxic granule components and ROS leading to
tissue destruction. Apoptosis can be partially restored by
the addition of anti-TNF antibody and IL-10 [118].
Macrophages show increased rates of apoptosis in
polymicrobial sepsis, associated with NO and caspase
activation, leading to loss of phagocytic function [119].
Cancer
There is evidence that a failure to initiate apoptosis
following DNA damage may cause cancer. Elevated levels
of c-myc are found in many tumours [120122]. In
human follicular lymphoma, translocation of chromosomes 14 and 18 causes up-regulation of Bcl-2 òncogene'
expression [48, 123]. Levels of Bcl-2 are elevated in
various other human cancers, for example lymphomas,
leukaemias, adenocarcinomas, renal and lung cancers,
neuroblastomas and melanomas [123126].
Other mutations may also be involved in carcinogenesis. A notable example involves the tumour suppressor p53 that represses Bcl-2 expression. The p53 gene is
deficient in over half of human cancers [127130]. For
example, in Wilm's tumour p53 mutations occur in
anaplastic areas.
Renal system
Embryological development of the kidney involves
periods of growth and apoptosis which are reflected by
the levels of Bcl-2 present [131]. Mice deficient in Bcl-2
develop polycystic kidney disease [132] whereas, Bcl-2
levels are high in all renal tumours [133].
Gastrointestinal system
Gastrointestinal diseases may be associated with excessive
or defective apoptosis. Shigella dysenteriae causes excessive
apoptosis of macrophages in the lamina propria of the
intestine by the release of IL-1b. Mice expressing a
mutant nonfunctional N-cadherin in intestinal villi
develop changes similar to Crohn's disease and show
increased rates of apoptosis in both villi and crypts, as well
as higher rates of adenomas.
Progressive inhibition of apoptosis appears to be
involved in the pathogenesis of gastrointestinal neoplasia,
in particular colorectal cancer [134]. Genes that regulate
apoptosis are mutant in colonic and gastric cancers. Wildtype p53, when introduced into human colon cancer cell
lines, inhibits cell growth and induces apoptosis [135].
However, p53 expression is associated with a poorer
prognosis in both colorectal and gastric cancers [4]
possibly owing to mutant or nonfunctional p53 that fails
to induce the usual apoptosis. The Bcl-2 protein can also
be detected in human cancers and is highest in adenomas
[136].
Hepatic cells develop apoptosis when infected with
viruses as in chronic hepatitis [137]. Abnormal activation
of cytotoxic T cells may be involved in human fulminant
hepatitis [95]. Paracetamol stimulates increases in intracellular calcium which activates Ca21-dependent nucleases
[138]. Apoptosis also appears to mediate allograft rejection
in a model of liver transplantation [139].
Reproductive system
Apoptosis is continually inhibited in many tissues of the
reproductive system owing to the presence of trophic
hormones from the pituitary, gonads and uterus. When
the hormones are removed, the tissues undergo atrophy.
Ovarian follicles undergo growth or atresia in response to
cyclic changes in luteinizing hormone and follicle
stimulating hormone; the endometrium, breast and
prostate are dependent on the steroid hormones and
regress when these are removed [140].
Therapeutic possibilities and future directions
The widespread involvement of apoptosis in the pathophysiology of disease lends itself to therapeutic intervention. In diseases caused by increased cell loss, such as
viral hepatitis and neurodegenerative disease, the aim will
1087

................................................................................................................................................................................................................................................
be to minimise apoptosis by modifying the signals which

trigger the response (e.g. Ca21, ROS) or interfering with
the effectors (e.g. caspases and endonucleases). However,
inhibition of apoptosis may be deleterious because new
tumours may arise when damaged cells are prevented
from committing suicide. In diseases caused by deficient
apoptosis, such as cancer, viral latency and autoimmunity,
methods of producing selective apoptosis are being
sought.
Agents targeting receptors or regulatory molecules and
agents targeting the final common pathway are attractive
possibilities. The soluble form of Fas could prove useful
for increasing apoptosis (e.g. in tumours). Antibodies to
Fas or Fas ligand may be useful in preventing apoptosis
(e.g. in neurodegenerative disease). Preliminary successes
in treating chronic inflammatory diseases, such as
rheumatoid arthritis and ulcerative colitis, with TNF-a
inhibitors have been reported, but these therapies have
proved disappointing in sepsis [141].
The regulatory molecule Bcl-2 may protect normal
cells from death induced by cytotoxic agents [142]. In
contrast, decreasing Bcl-2 in cancer cells can reinstate
chemo- and radio-sensitivity. This can be achieved by
treatment with synthetic short DNA single strands called
antisense oligonucleotides, which bind to specific messenger RNA sequences and prevent production of the
offending protein [143].
The caspases are of interest from a therapeutic point of
view as specific inhibitors exist. Tetrapeptide aldehydes
are potent inhibitors of ICE but are toxic [4]. They act by
specifically binding to the protease and preventing
cleavage of the target proteins. Inhibitors of ICE can
inhibit apoptosis in a number of cell systems. They may
have a role in sepsis and chronic inflammatory and
neurodegenerative disease [144].
Cancer
Kerr et al. [145] found that anticancer agents induce
apoptosis in tumours. Chemotherapeutic agents reported
to induce apoptosis include the alkylating agents (cyclophosphamide, mitomycin C, nitrogen mustard), topoisomerase II inhibitors (daunorubicin, adriamycin),
dexamethasone, antimetabolites (methotrexate, 5-fluorouracil, 5-azacytidine), cisplatin, microtubule disrupters
(vincristine, vinblastine, taxol), cycloheximide, bleomycin,
cisplatin, tamoxifen and cytosine arabinose [30, 90].
Irradiation and cytotoxic agents produce DNA damage
which predisposes to p53 enhancement of apoptosis. If
p53 is defective, then resistance to chemotherapy may
result [129]. The activation of the p53 pathway in neoplasms to regain chemosensitivity is a potentially powerful
therapeutic tool that can render the tumour apoptotic.
This may be possible through p53 gene-specific therapy.
1088
Such therapy has been attempted using retrovirally

introduced wild-type p53 on non-small cell-lung cancer
with encouraging results [129]. Nicotine has been shown
to suppress apoptosis in lung cancer in humans [146].
Inflammatory disease
Corticosteroids induce eosinophil apoptosis but inhibit
neutrophil apoptosis. The treatment of asthmatic patients
with corticosteroids can cause eosinophil death and
macrophage engulfment [147]. The detection of this
process in airway secretions of asthmatic patients is
associated with clinical improvement [148].
Ischaemia and reperfusion
Apoptosis has been demonstrated in areas of brief
ischaemia and reperfusion in the brain, heart, liver and
kidney [11, 82]. Further understanding of the molecular
mechanisms should allow novel protective strategies to be
developed.
Gastrointestinal tract
Cytotoxic drugs induce apoptosis in studies of human
gastrointestinal cancer cells as well as normal mouse
intestine, which may account for their therapeutic action.
Chronic ingestion of nonsteroidal anti-inflammatory
drugs may be useful in preventing colonic cancer, possibly
by induction of apoptosis. Cyclo-oxygenase-2 (COX-2)
may enhance formation of cancer by changes in cellular
adhesion and by inhibition of apoptosis via enhanced
Bcl-2 expression [4, 134]. Non-steroidal anti-inflammatory drugs, which inhibit COX-1 and COX-2, can
prevent up-regulation of Bcl-2 by prostaglandins and
prevent colorectal carcinoma. The protective effects of
dietary fibre may be via its fermentation by bacteria in the
colon to form short-chain fatty acids (e.g. butyrate) which
promote apoptosis [4].
Conclusion
Cell injury, as a result of physical, biochemical or

biological insults, or from a deficiency of vital substances,
induces expression of adaptive stress response genes. The
interactions of the acute phase, heat shock and oxidative
stress responses appear to determine the fate of the cell.
The intracellular response to injury leads to distinct
genetic expression that mediates the cellular changes
which are usually specific to cell type and injury. These
cellular responses are usually cytoprotective but can
precipitate apoptosis. Reducing agents, antioxidants,
anti-TNF antibodies, steroid antagonists and inhibitors
of protein synthesis can modulate apoptosis. Novel genedirected treatment for various disease states, such as
multiple organ dysfunction syndrome and some cancers,

................................................................................................................................................................................................................................................
may be available as the understanding of the impact and

mechanism of stress gene responses to injury are
elucidated.
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Anaesthesia, 2000, 55, pages 10811093