J Clin Pathol: Mol Pathol 2000;53:55–63 55

Reviews

Demystified . . .

Apoptosis

S AVord, S Randhawa

Abstract By 1887, others were investigating the diVer-

Apoptosis is the genetically regulated ent morphologies of cell death.2 In 1885,
form of cell death that permits the safe Walther Flemming was studying the regression
disposal of cells at the point in time when of epithelium in mammalian lymphoid folli-
they have fulfilled their intended biologi- cles, and it was he who produced the first
cal function. Examples of apoptosis can be drawings of what we today would recognise
cited throughout the whole of the animal morphologically as apoptosis, and he adopted
and plant kingdoms. It is a vitally impor- another term “chromatolysis”.3 The first re-
tant process during normal development view describing chromatolysis appeared in
and the adult life of many living organ- 1914 and was written by Ludwig Graper, a
isms. In humans, dysregulation of apopto- German anatomist studying yolk sac
sis can result in inflammatory, malignant, shrinkage.4 Almost 40 years later, in 1951
autoimmune, and neurodegenerative dis- Glucksmann was able to describe “chroma-
eases. In addition, infectious agents, in- tolysis” during embryo development.5 Thirteen
cluding viruses, exploit cellular apoptosis years hence, experimental pathologists began
in the host to evade the immune system. in earnest to investigate the ways in which the
This review gives a brief historical per- cells in individual organs die during develop-
spective of some of the landmark discov- ment and in response to insult or injury.6
eries in apoptosis research. The Some of the early studies of this process in
morphological and biochemical stages of mammalian tissues centred around the cellular
apoptosis are then covered, followed by an pathology of liver atrophy after restriction of
overview of how it can be studied in the the blood supply by portal vein ligation. Even
laboratory. Finally, the implications for then, the liver was recognised as a potentially
therapeutic intervention in disease treat- useful organ in this field of study because of its
ment are discussed. phenomenal capacity to regenerate.6 7 It was
(J Clin Pathol: Mol Pathol 2000;53:55–63) essentially from this model that the concept
was evolved whereby cell populations could be
Keywords: apoptosis; necrosis; tumour necrosis factor; held in balance not only by proliferation but
development also by some type of controlled death. Even at
that time, many good theoretical reasons for
such a mechanism were obvious including,
Slime mould, nematode worms, butterfly regeneration and repair, maintenance of cellu-
The Liver Research larvae, fruit flies, dwarf tree frogs, chickens, lar homeostasis, and control of organ size.
Laboratories and MRC
Centre for Immune
mice, and rats, to name but a few species, have Concepts relating to genetic editing for the
Regulation, University all played their part in defining the importance deletion of defective, mutated, infected, or oth-
of Birmingham of “apoptosis” or “programmed cell death”. erwise functionally eVete cells were shortly to
Institute of Clinical Apoptosis refers to the genetically regulated follow, and the scientific understanding of
Science, Queen form of cell suicide that is morphologically dis- regulated cell death slowly began to advance.7 8
Elizabeth Hospital, tinct from the degenerate process of necrosis. Several early studies focused on histopatho-
Edgbaston,
Birmingham B15 2TH, In 1858, Virchow described the progressive logical observations, and during the mid to late
UK changes in the appearance of body tissues that 1960s, a great deal was learnt about cell death
S AVord occur shortly after death, and it was he who was at the ultrastructural level using electron
S Randhawa the first to recognise that more than one form microscopy.6 In 1972, a group of pathologists
of cell death existed. Necrosis being where “the in Edinburgh—Kerr, Wyllie, and Currie—were
Correspondence to:
Dr AVord mortified cell is left in its external form” and working in the field and realised that conven-
email: s.c.aVord@ “necrobiosis or shrinkage necrosis being where tional terms such as necrobiosis, shrinkage
bham.ac.uk the cell vanishes and can no longer be seen in necrosis, chromatolysis, coagulative necrosis,
Accepted for publication its previous form”.1 Necrobiosis is what we or ischaemic death were confusing and inad-
21 December 1999 now recognise as apoptosis. equate to delineate the two forms of cell
56 AVord, Randhawa

death.8–10 Thus, it was Kerr, Wyllie, and Currie Apoptosis or necrosis: are there really
who proposed, with the assistance of Professor two pathways to death?
James Cormack (at that time the Professor of It is important to recognise that these terms
Greek at Aberdeen University) the adoption of relate to a “morphological description” of cell
an unambiguous term “apoptosis” to describe death only, and it remains possible that the two
specifically one of these methods of cell death.8 processes might be part of a continuum, with
Even today, it seems that pathologists are morphological necrosis being the ultimate fate
divided in opinion about the term “necrotic even for cells that undergo apoptotic changes
cell death”, and alternative terminology has initially.15
been proposed recently.11 That the two pathways overlap to some
It was perhaps also Kerr, Wyllie, and Currie extent is evident because in vivo one type of cell
who were the first to realise the far reaching death is often seen accompanying the other and
implications of apoptotic cell death within the in cultured cells apoptotic cells can, under cer-
tain circumstances, assume necrotic
context of human cell biology and disease.
morphology.15
Their work in the early 1970s has proved to be
It is also important in the broader context to
a scientific landmark, which to a large extent
appreciate that regulated cell death occurs
still upholds the principles that have conceptu- across the entire animal and plant kingdom—
ally dominated research in this field until the but for diVerent reasons in humans compared
present day.8–12 with the slime mould (dichtyostelium).16 17 It
Early histopathological studies gave enough therefore follows that during the course of evo-
information to describe the division of apopto- lution, many diVerent pathways of achieving
sis into discrete morphological stages, and to the same endpoint have developed. That is to
advance hypotheses that have since been say that cellular dismantling and recycling has
substantiated and underpinned by research developed in response to environmental pres-
performed in laboratories throughout the sures faced by the individual organism. In
world. During the 1970s, however, it is fair to addition, it should not always be assumed that
say that interest in apoptosis research reached a necrosis has no part to play throughout the
plateau, with many members of the scientific normal life cycle of living organisms. For
community being unimpressed and uncertain example, trees use dead tissues as an important
of the potential importance of apoptosis. structural component. In the dichtyostelium
The real watershed for apoptosis research slime mould, stalk cell replication, a crucial
did not arrive until the early 1080s, when a phase leading to spore dispersal, is arrested via
succession of papers led to a huge revival of necrosis rather than apoptosis.16 Some very
interest. In 1980, Andrew Wyllie produced a recent and fascinating developmental studies
paper on glucocorticoid induced apoptosis in also suggest that when the apoptotic pathway is
cultured lymphocytes, which was possibly genetically deleted, the normal developmental
responsible for the resurgence of interest.13 cell loss during mouse forelimb formation can
Three years later, apoptosis was shown to be still proceed—and it does so predominantly by
linked to the activation of an endonuclease that necrosis.18 Thus, an open mind is still required
cleaved DNA into regular sized fragments, about the relative contribution of these two
which could be demonstrated by gel electro- types of cell death to development and cellular
phoresis. Thus, the first biochemical evidence homeostasis.
for apoptotic cell death was established.14 Although we still have an incomplete picture
From that point forward, there has been of the individually tailored mechanisms of
almost a decade and a half where we have apoptosis or “programmed cell death”, there is
learned much about the detail of the remark- now a broad acceptance of its importance to all
able and genetically complex process of living organisms. SuYcient genetic and bio-
apoptosis. Powerful genetic manipulation and chemical evidence exists to show that the path-
way for regulated cell death in digit formation
molecular biology techniques have led to an
in the developing mouse embryo—for exam-
exponential growth in our knowledge of apop-
ple, is diVerent to the pathway that has evolved
tosis, and we now have a good, albeit
for the modulation of total organ mass or
incomplete, understanding of the myriad of leucocyte numbers, the maintenance of
genes that are involved in the regulation of this immunological tolerance, the protection
process. The race is now on to dissect these against deleterious chromosomal aberrations/
complex interactive pathways, in the hope that mutations, or the elimination of pathogenically
more detailed knowledge will lead to the design infected cells within the whole organism.
of more specific strategies for the combat of In the course of this review we hope to cover
disease. in summary what is known about how apopto-
Today, a current literature search using sis is initiated, how it is regulated, and what can
apoptosis as a keyword will reveal in excess of happen when it goes wrong, all within the con-
29 200 listed manuscripts, and interest in text of the pathophysiological processes that
apoptosis research shows no signs of abating. occur throughout life in humans.
As new apoptotic genes continue to be identi-
fied, and new apoptosis related functions are The stages of apoptotic cell death
assigned to familiar proteins, our understand- Apoptosis can be thought of as a series of pro-
ing of this complex process will have to remain gressive steps that lead desirably to the eYcient
under constant review for the foreseeable dismantling of the cell in a manner that
future. minimises the risk of leakage of potentially
Demystified . . . Apoptosis 57

harmful intracellular contents into the sur- leaves the bone marrow and enters the periph-
rounding microenvironment, where it could eral circulation, it is terminally diVerentiated
otherwise have harmful cytotoxic side eVects and incapable of further division. Should it not
(fig 1). be recruited to an inflammatory lesion (where-
upon it would release its potent and highly
STAGE 1: TRIGGERING APOPTOSIS—MECHANISMS cytotoxic contents via degranulation), it se-
OF STIMULATION quester in the lung, where the senescent
It seems likely that all living cells have evolved neutrophils undergo apoptosis. Here, they are
the genetic capability to undergo apoptosis normally phagocytosed by resident alveolar
spontaneously. Although the mechanisms that macrophages.21–23 This mechanism ensures the
lead to the starting of the “biological clock” are safe disposal of these otherwise harmful cells.
not understood, it seems likely that alterations When this system is stressed as a result of over-
in the environmental conditions that the cell whelming toxic injury or infection, massive
experiences can start, accelerate, or slow down systemic damage results (septic shock syn-
the process.19 20 drome).
To enter into the apoptotic cycle the cell first In the lymph nodes, cells that are not
encounters a signal to activate the relevant exposed to frequent antigenic stimulation
genetic machinery. A good example of physi- undergo clonal deletion via apoptosis. This is a
ologically important apoptosis in adult life is very good example of how withdrawal of
the life cycle of the mature neutrophil. When it factors can lead to apoptotic cell death.24 This

Figure 1 The stages of apoptosis. This diagram illustrates the four basic stages of the apoptotic pathway. Once the cell has
reached stage two and the caspase pathway has been activated it is believed that the process is irreversible and the cell
cannot be rescued. After stage three, if the cell is in close enough proximity to a phagocytic neighbour and is displaying the
right molecular signature it is engulfed and broken down within the phagocyte. If the apoptotic cell is not recognised it will
eventually assume necrotic morphology, so called “secondary necrosis”.
58 AVord, Randhawa

mechanism is of crucial importance in the rec- with and activate other apoptosis inducing
ognition of self and non-self, and maintenance receptor–ligand pairs. In addition, it seems that
of immunological tolerance.25 Breakdown of some family members, which are incapable of
this regulatory pathway results in a range of signalling because they lack a cytoplasmic tail,
autoimmune and malignant conditions.26 can function as “decoy receptors”. At present,
There is now also good evidence that entry into little is known about the true biological role of
the proliferative cycle is a crucial step in sensi- many of these receptor family members,
tising some, but not all, cells to apoptotic beyond their sequence homology and distribu-
signals.26 tion throughout tissues. Detailed discussion of
There are stimuli that originate from outside individual receptors is clearly beyond the scope
the cell that can advance or delay apoptotic of this review, and the reader is referred to
death. This stage of the process can be other articles for further information.34–36
triggered by agents that can penetrate the cell Expression of some of the TNF receptors
directly, and modulate the apoptotic cascade in appears to be restricted to specific cell types
the absence of specific cell surface receptors. whereas others (for example, Fas (CD95/Apo
Examples of such agents include heat shock/ 1)) are distributed across a wide range of cell
stress factors,25 free radicals,27 ultraviolet types, and are thought to have a central role in
radiation,28 numerous drugs and synthetic the triggering of apoptosis.37–39 This prototypic
peptides,29 toxins,30 and potent lymphocyte family member illustrates how many receptor
enzymes (the granzymes).31 Other mechanisms mediated apoptotic pathways can be activated.
are dependant on expression of appropriate cell
surface receptors. STAGE 2: INTRACELLULAR SIGNALLING AFTER
Receptor mediated apoptosis has been dem- RECEPTOR LIGAND BINDING
onstrated for numerous growth factors (for The TNF receptor superfamily members are
example, transforming growth factor â32 and all typical type 1 membrane spanning glycopro-
cytokines, including tumour necrosis factor teins, with the N-terminus on the outside of the
(TNF)).33 Of these molecular families, the cell containing anywhere between one and six
TNF like molecules are a family of ligands that ligand binding domains. Within the cell, the
exist as soluble or cell bound forms and which C-terminus typically (for the death transducing
are known to have a major role in the modula- receptors see fig 2) contains a region of about
tion of cell survival/apoptosis in cells of many 60–70 residues, which upon activation with a
lineages.34 35 The TNFs act via a large family of trimeric ligand, forms a cluster and a complex
receptors expressed on the surface of the target beneath the cell membrane with a number of
cell, known as the TNF receptor superfamily. cytosolic proteins to form the active “death
The TNF receptor family comprises at least 19 domain” (for example, Fas forms the Fas asso-
members, which are grouped together based ciated death domain (FADD)). This receptor–
on structural homology, but which in reality ligand complex then becomes the recognition
have a wide array of biological eVects that molecule for a precursor enzyme, procaspase
sometimes extend beyond the regulation of 8.40
apoptosis.35 36 At least four of them can trigger The caspase family of enzymes contains
apoptosis directly, and although not containing cysteine proteinases that mediate a whole range
an intracellular death domain themselves, of intracellular events, and a number of agents
several others do have the potential to interact act by modulating caspase expression and
activity directly, bypassing the receptor
pathway41 (table 1). There are presently 13
known caspase family members, which form an
intracellular proteolytic cascade that modulates
many cellular events in the apoptotic pathway,
including activation of transcription factors.42
Transcription factors are regulatory proteins
that bind to specific initiation sites on DNA,
and in turn modulate expression of genes that
regulate production of pro-apoptotic and
antiapoptotic factors. Such important apop-
totic genes include the bcl-2 family of mito-
chondrial proteins,43 cytochrome c,44 the
tumour suppressor genes p53 and p21(WAF
1), and many others.45 In a highly complex and
as yet poorly understood way, it is the balance
between these pro-apoptotic and antiapoptotic
factors that controls the fate of the cell (table
1).46
There is recent evidence suggesting that not
all apoptotic cell death requires caspase activa-
tion. However, little is currently known about
Figure 2 Apoptotic cell death mediated by the Fas receptor pathway. EVector cells (for caspase independent death and its pathophysi-
example, activated T lymphocytes) present the Fas ligand (FasL) in a conformation that ological relevance awaits clarification.18
permits crosslinking and aggregation of the Fas receptor on the target cell. The clustering of Much of what is currently known about the
Fas is the first step in activating the intracellular apoptotic cascade. Formation of a
complex, the Fas associated death domain (FADD), then results in binding and activation molecular and genetic regulation of apoptosis
of procaspase 8 and the caspase cascade. has been the result of work carried out on the
Demystified . . . Apoptosis 59

Table 1 Genes and molecules that regulate apoptosis

Gene/molecule EVects

Bcl-2 subfamily (Ced9 in Caenorhabditis elegans)

Bcl-2 Promotes survival
Bcl-xl Promotes survival
Bcl-w Promotes survival
Bcl-xs Promotes death
Bax family
Bax Promotes death
Bak Promotes death
Bok Promotes death
BH3 subfamily
Bad Promotes death
Bik Promotes death
Bid Promotes death
Blk Promotes death
HRK Promotes death
BimL Promotes death
Egl (C elegans) Promotes death
Caspases 1–13* (Ced3 in C elegans) These interleukin 1â converting enzyme (ICE) like cysteine
proteinases form a central part of the apoptotic cascade
Apaf-1 (Ced4 in C elegans) These adaptor molecules link caspase (Ced3) activation and
Bcl-2 (Ced9) expression. Their functions in promoting death
or survival remain to be defined
p53 family of tumour supressor genes p53 is necessary for apoptosis induced by agents that cause DNA
damage. Growth arrest occurs by activation of p21. p53 can
also aVect the expression of bcl-2 and bax directly. p21/WAF1
inhibits cyclin dependent kinases and prevents progression
through the cell cycle
Nitric oxide This molecule prevents apoptosis by altering bcl-2 expression
and by nitrosylation of caspases
Cytochrome c This molecule induces apoptosis via caspase activation

This table summarises the major families of regulatory genes that are activated when a cell undergoes apoptosis. The list is by no
means exhaustive but gives representative family members.
The products of the bcl-2 gene subfamily are mitochondrial proteins that can exist as homodimers or heterodimers made up of dif-
ferent pairings. Their functions are still unclear but changes in the ratio of their expression has profound eVects on cell survival.
*Inhibitors of ICE have also been described recently.

nematode worm, Caenorhabditis elegans, where leading to the exposure of anionic phospholip-
the Ced (cell death abnormal) and Egl gene ids and phosphatidylserine (PS). These are
family members have been cloned and se- thought to be the molecular signals recognised
quenced, and to which an array of apoptotic by neighbouring phagocytic cells.51 Eventually,
functions have been ascribed. Because many of the plasma membrane of the cell begins to bud
these genes are highly conserved, mammalian oV encapsulating and packaging lysosomes,
homologues of the Ced genes have now been mitochondria, chromatin fragments (known
found (table 1).46 also as Councilman bodies), and other degen-
erate organelles including the Golgi/
STAGE 3: APOPTOSIS—THE MORPHOLOGICAL AND endoplasmic reticulum (figs 1 and 3).
BIOCHEMICAL CHANGES
Release of intracellular calcium and depletion STAGE 4: PHAGOCYTIC RECOGNITION OF
of ATP accompany the ongoing process.47 In APOPTOTIC BODIES
contrast to necrosis, there is no mitochondrial The importance of this part of the process to
swelling and the cell begins to shrink with the normal tissue homeostasis and during the
plasma membrane remaining intact. Chroma- resolution of the inflammatory response is now
tin begins to condense and marginate towards well recognised.51 52 Hypothetically, defects in
the nuclear membrane periphery (fig 1). Mito-
chondrial activity increases with the synthesis
and release of apoptosis regulatory proteins
such as the bcl-2 family members, cytochrome
c, and nitric oxide.47 The activated caspase cas-
cade continues to initiate the induction of other
apoptotic genes, including death promoters
such as p53 or p21.
Mutations in one of these tumour suppressor
genes (p53), in particular, is thought account
for a large proportion of malignancies in
human disease,26 and p53 is a major target for
transforming viral oncoproteins.48 Other im-
portant pro-apoptotic genes include those that
encode the endonuclease that degrades the
DNA into 50 kb fragments, the biochemical
hallmark of apoptosis in most, although not all,
cell types.49 Genes are also activated that lead to
expression of other cell surface phagocyte rec- Figure 3 The morphology of apoptosis. This
ognition molecules, such as intercellular adhe- photomicrograph shows a culture of promonocytic U937
cells, some of which show classic morphological features of
sion molecule 3 (ICAM 3).50 A crucial step apoptosis (arrow A). The cell marked N shows the normal
appears to be the loss of membrane asymmetry, morphology of a viable neighbour for comparison.
60 AVord, Randhawa

phagocyte recognition of apoptotic cells may The fact that the vitronectin receptor and
lead to persistence of inflammatory disease, CD36 can signal via tyrosine kinase implies
which is exacerbated because apoptotic cells that these molecules in particular might be of
eventually assume necrotic morphology (so crucial importance in the subsequent activa-
called secondary necrosis). This idea has tion of the signalling pathway in the phagocytic
gained credence because it seems a particularly cell, about which virtually nothing is currently
attractive explanation for persistent inflamma- known.
tion and tissue damage. As yet, there is little
histopathological or clinical proof that such a How can apoptosis be studied?
mechanism occurs in vivo, but this field of work MOLECULAR GENETICS
is continuing to advance. Today’s apoptotic research is becoming domi-
The point at which apoptotic cells are recog- nated be the power of modern molecular biol-
nised by phagocytic cells is not really known, ogy; in particular, the use of transgenic animal
although it is probably at quite an early stage, models to test the function of particular genes.
because in tissues where cell turnover is high, There are well known caveats to these models,
the frequency of mature apoptotic bodies can the most important of which is that, as the
be relatively low.53 Research into the mecha- number of interactive gene products in any
nisms involved in phagocytic recognition are particular system increases, so does the likeli-
still at an early stage. Several molecules on the hood of redundancy. In other words, when a
surface of the phagocyte and the apoptotic cell gene is deleted and no biological eVect is
have been identified as important mediators of observed it is possible that other pathways have
the process (fig 4), most of which have turned compensated for the defect. That being said,
out to be cell surface molecules that have been transgenics do continue to be a powerful
familiar to immunologists and biochemists for analytical approach to the analysis of gene
function. In relation to apoptotic cell death,
quite a few years.54–56 Research in this area is
transgenic animals where death receptor genes,
currently focused on the professional macro-
including Fas and CD40 or their cognate
phages of leucocytic origin, and much less is
ligand encoding genes, have been deleted have
known about phagocytosis of apoptotic cells in
given important clues as to the importance of
other systems. Key molecules on the phagocyte
these molecules for the regulation of apoptosis
include the class A scavenger receptors macro- in several cell types.57 58 The Fas ligand
sialin and ABC 1, the vitronectin receptor, and “knockout animals” suVer from lymphoprolif-
the class B scavenger receptors CD36 and erative disease and liver hyperplasia. CD40–
CD14 (a glycophosphatidylinositol anchored ligand knockout animals are also severely
protein which is the lipopolysaccharide recep- immunocompromised. Interestingly, CD40–
tor). These molecules interact with several ligand knockout animals are also susceptible to
counter-receptors on the apoptotic cell includ- the development of cholangiocarcinoma, have
ing anionic phospholipids, PS, and ICAM 3. liver hyperplasia, and fail to clear virally
On the phagocytic cell, the vitronectin receptor infected cells eVectively.59 60 All these observa-
(ávâ3 integrin) and CD36 both have the ability tions underpin the systemic importance of an
to bind a bridging molecule thrombospondin. eVective functional apoptotic pathway .
Although the partner for thrombospondin In addition, the ability to modify individual
bridging on the apoptotic cell has yet to be cell types, either by gene deletion or inducing
defined, likely candidates are PS and ICAM 3, or inserting pro-apoptotic and antiapoptotic
although the latter has been shown recently to genes, has contributed greatly to our under-
partner CD14.50 53 standing of the role of individual molecules in
apoptosis. For example, manipulating hepato-
cytes to overexpress the survival factor gene
bcl-2 makes the cells resistant to Fas mediated
apoptosis.61 Molecular analysis of apoptotic
gene mutations has also provided some tanta-
lising clues about how malignant transforma-
tion can occur. Aggressive tumour growth has
been associated with mutations or the aberrant
expression of several apoptotic genes, including
bcl-2 family members, p53, Fas,62 and CD40.63
An understanding of how these genes function
oVers great potential for therapeutic interven-
tion.

CELL AND MOLECULAR BIOLOGY

A great deal of information can also be gained
from cell biology, molecular biology, and
biochemical approaches to the study of cells in
Figure 4 Phagocytic recognition of an apoptotic cell. At present, little is known about the tissue culture, or ex vivo in tissue sections.
interactions between apoptotic cells and phagocytes, but a series of molecules seem to be
important including phosphatidylserine (PS), anionic phospholipids, and intracellular These approaches fall into categories that
adhesion molecule 3 (ICAM 3). The hierarchical nature of these interactions is not yet relate to either the morphological or biochemi-
known but recent evidence suggests that ICAM 3 partners CD14 on the macrophage. An cal assessment of apoptosis. There are no single
important pairing also seems to be between PS and the vitronectin receptor or CD36. This
probably occurs via thrombospondin, which can avidly bind both molecules on the specific markers of this process, and it is widely
phagocyte. accepted that several diVerent techniques
Demystified . . . Apoptosis 61

should be carried out. As stand alone tech- dyl transferase d uridine triphosphate nick end
niques go, the gold standard method for labelling (TUNEL) method.66 Both techniques
morphological assessment is electron micros- exploit the endonuclease dependant cleavage
copy, where the ultrastructural changes that of DNA. The assumption is made that this
typify apoptosis can be seen in individual occurs with much higher frequency in apop-
cells.64 totic cells than under any other circumstances.
Quantification of apoptosis requires other Both techniques rely on the labelling of the
approaches. Electrophoretic analysis of DNA, nicked DNA with a chromogen or fluorogen
the so called “DNA laddering technique”, conjugated complex.
allows confirmation of the presence of de- Other techniques exploit the changes in
graded DNA, but in most cases does not nuclear morphology and cell membrane bio-
permit quantitative assessment of the degree of chemistry, and in some circumstances allow for
apoptosis (fig 5). This method is only really the automated measurement of apoptosis via
appropriate for isolated pure populations of flow cytometry. These methods rely on various
cells. Assessing cell and nuclear morphology combinations of propidium iodide, which can
microscopically in combination with an enter necrotic cells and stain the nucleus, and
immunohistochemical technique for detection labelling of the cell surface phosphatidyl serine
of DNA strand breaks (which occur at high residues, which are expressed at early stages of
frequency in apoptotic cells) is a widely used apoptosis, with an anticoagulant protein called
and generally accepted approach. annexin V, conjugated to a reporter molecule,
There are many apoptosis detection kits now such as fluorescein or biotin (fig 7).
commercially available that are based on DNA The downstream intracellular events of
fragmentation, most being variations on similar apoptosis can also be assessed using a variety of
themes. Two frequently used techniques are cell and molecular biological approaches.
the in situ DNA end labelling method (ISEL)65 Again an abundance of commercially available
(fig 6) and the in situ terminal deoxynucleoti- reagents are readily available. Gene expression
and the consequent protein synthesis can be
monitored using western immunoblotting (for
proteins) and reverse transcriptase PCR (for
mRNAs). A wide range of antibodies to recep-
tors, ligands, transcription factors, caspases,
and tumour supressor gene products permits
localisation of many molecules using immuno-
histochemistry. There are also several chro-
23 130 mogenic substrates, with varying degrees of
9416 specificity for the caspase family members,
6557 which allow for functional assessment of
4361 enzyme activity.

The implications of apoptosis in disease

2322 and potential for treatment
2027 The consequences of manipulating apoptosis
are potentially beneficial and deleterious.
Although we still have much to learn, we
already know that the system is extremely
complex, and there are several areas where the
modulation of apoptotic death could be of
benefit in the treatment of disease in the future.
(1) Control of malignant disease: the induc-
tion of apoptosis in malignant cells would
be desirable.
(2) Delay of premature senescence/
563 neurodegenerative disorders: the preven-
tion or delay of apoptosis in neural cells is
a desirable goal.
(3) Regulation of inflammatory disease: the
induction of apoptosis and phagocytosis
would be desirable for the suppression/and
resolution of the inflammatory response.
(4) Treatment of transplant rejection: the pre-
vention of apoptosis in parenchymal cells
and the induction of apoptosis for estab-
lishing immunological tolerance (cells of
the immune system) would both be desir-
Figure 5 Electrophoretic analysis of DNA from apoptotic cells. DNA extracts can be able in this setting.
purified, run on an agarose gel, and stained with fluorescent ethidium bromide. DNA from (5) Regulation of tissue regeneration/repair:
apoptotic cells (aged blood polymorphs in this case) forms a distinctive laddering pattern as induction of apoptosis would be useful to
a result of endonuclease cleavage (lane 1). Lane 2, DNA isolated from necrotic cells; lanes
and 6, no sample; lanes 3 and 4, DNA immediately after isolation (before treatment); lane limit fibroblast activity and control scar
7, standard DNA molecular weight markers. Band sizes are in kDa. formation.
62 AVord, Randhawa

sued vigorously worldwide. It remains to be

seen which, if any, approach will be suYciently
successful to oVer real hope for therapeutic use
in the treatment of a disease where dysregula-
tion of cell death is implicated.

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