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2013

Apoptosis
Its Significance in Cancer and Cancer Therapy
John F. R. Kerr, Ph.D.,* Clay M. Winterford, Assoc.Dipl.Appl.Biol.,*
and Brian V. Harmon, Ph.D.t

Apoptosis is a distinct mode of cell death that is responsible for deletion of cells in normal tissues; it also
occurs in specific pathologic contexts. Morphologically,
it involves rapid condensation and budding of the cell,
with the formation of membrane-enclosed apoptotic bodies containing well-preserved organelles, which are
phagocytosed and digested by nearby resident cells.
There is no associated inflammation. A characteristic biochemical feature of the process is double-strand cleavage
of nuclear DNA at the linker regions between nucleosomes leading to the production of oligonucleosomal
fragments. In many, although not all of the circumstances in which apoptosis occurs, it is suppressed by
inhibitors of messenger RNA and protein synthesis.
Apoptosis occurs spontaneously in malignant tumors, often markedly retarding their growth, and it is increased
in tumors responding to irradiation, cytotoxic chemotherapy, heating and hormone ablation. However, much of
the current interest in the process stems from the discovery that it can be regulated by certain proto-oncogenes
and the p53 tumor suppressor gene. Thus, c-myc expression has been shown to be involved in the initiation of
apoptosis in some situations, and bcl-2 has emerged as a
new type of proto-oncogene that inhibits apoptosis,
rather than stimulating mitosis. In p53-negative tumorderived cell lines transfected with wild-type p53, induction of the gene has, in rare cases, been found to cause
extensive apoptosis, instead of growth arrest. Finally, the
demonstration that antibodies against a cell-surface protein designated APO-1 or Fas can enhance apoptosis in
some human lymphoid cell lines may have therapeutic
implications. Cancer 1994; 73:2013-26.
From the 'Department of Pathology, University of Queensland
Medical School, Herston, Queensland; and the tSchool of Life
Science, Queensland University of Technology, Brisbane, Queensland, Australia.
Supported by the Queensland Cancer Fund and the University
of Queensland.
Address for reprints: John F. R. Kerr, Ph.D., Department of Pathology, University of Queensland Medical School, Herston, Brisbane, Queensland 4006, Australia.
Accepted for publication November 24, 1993.

Key words: apoptosis, programmed cell death, cell deletion, DNA fragmentation, radiation, anti-cancer drugs,
hyperthermia, hormone ablation, proto-oncogene, tumor
suppressor gene.

Oncologists traditionally have been concerned primarily with cell proliferation. However, apoptosis (the distinctive form of cell death that complements cell proliferation in normal tissue homeostasis)' increasingly has
been attracting their attention.'f3
The realization that apoptosis occurs in tumors is
not new. More than 20 years ago it was suggested that
apoptosis may account for much of the spontaneous
cell loss known from kinetic studies to occur in many
and it has been clear for some time that its
extent often is enhanced in tumors by well-established
treatment modalities, such as
cytotoxic
chern~therapy:-'~ heating,'0~"~'6-'8and hormone ablation.l9-'' However, during the past few years, advances
in understanding of the control of apoptosis at the molecular level have extended its potential oncologicsignif icance far beyond the mere provision of a mechanistic
explanation for tumor cell deletion. In particular, the
discovery that apoptosis can be regulated by the products of certain proto-oncogenes and the p53 tumor suppressor
has opened up exciting avenues for
future r e ~ e a r c h . ~
The proposition that apoptosis is a discrete phenomenon that is fundamentally different from degenerative
cell death or necrosis is based on its morphology, biochemistry, and i n c i d e n ~ e . In
~ ~this
~ ~article,
~ ~ - ~we
~ de-.
scribe these, placing special emphasis on cancer. We
also review the results of recent work on regulation of
the process and discuss the oncologic implications of
this new knowledge.
Morphology of Apoptosis

The description that follows is based on our studies and


the published reports of others. We will not justify each

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Figure 1. Diagram illustrating sequence of ultrastructural changes in


apoptosis (2-6) and necrosis (7 and 8). (1) Normal cell. Early
apoptosis (2) is characterized by compaction and margination of
nuclear chromatin, condensation of cytoplasm, and convolution of
nuclear and cell outlines. (3) At a later stage, the nucleus fragments,
and protuberances that form on the cell surface separate to produce
apoptotic bodies, which (4) are phagocytosed by nearby cells and
(5 and 6) degraded within lysosomes. (7) The development of
necrosis is associated with irregular clumping of chromatin, marked
swelling of organelles and focal disruption of membranes. (8)
Membranes subsequently disintegrate, but the cell usually retains
its overall shape until removed by mononuclear phagocytes.

Figure 2. Apoptosis of murine NS-I myeloma cell occurring


spontaneously in culture. Note the sharply delineated masses of
condensed chromatin in membrane-enclosed nuclear fragments and
remnant of nucleolus (arrow). Ribosomes are well preserved
(electron micrograph, original magnification X 17,500).

(Figs. 3 and 4) to produce membrane-bounded apoptotic bodies (Fig. 5). The size and composition of the latter
vary considerably; many contain several nuclear fragments (Fig. 5) whereas others lack a nuclear component. In addition, the extent of the nuclear and cellular
budding vanes with cell type, often being relatively restricted in small cells with a high nucleocytoplasmic

statement with lists of references but will give a composite overview; supporting bibliographies and many additional illustrations can be found in comprehensivereviews.41.53,54

The contrasting ultrastructural features of apoptosis and necrosis are shown in stylized form in Figure 1.
Apoptosis characteristically affects scattered single
cells, not groups of adjoining cells, as is the case with
necrosis. The earliest recognized morphologic changes
are compaction and segregation of the nuclear chromatin,with the formation of sharply delineated, uniformly
finely granular masses that become marginated against
the nuclear envelope, and condensation of the cytoplasm. Progression of the condensation is accompanied
ofnthe nuclear and cell outlines, and this
by c o n v o ~ u ~ o
is
by breaking up Of the
into discrete
fragments that are surrounded by a double-layered envelope (Fig. 2) and by budding of the cell as a whole

Figure 3. Same culture as that illustrated in Figure 2. Separation of


surface protuberances is leading to apoptotic body formation. Some
nuclear fragments show peripheral masses of condensed chromatin,
whereas others are uniformly dense in plane of section (electron
micrograph, original magnification X13,900).

Apoptosis in Cancer/Kerr et al.

Figure 4. Apoptosis in murine EMT6 mammary tumor growing in


muscle 2 hours after heating at 44C for 30 minutes. Note the
marked condensation of cytoplasm with preservation of integrity of
organelles, nuclear fragmentation, and budding of cell to form
apoptotic bodies (electron micrograph, original magnification
X 13,600).

ratio, such as lymphocytes. The cytoplasmic organelles


of newly formed apoptotic bodies remain well preserved (Figs. 4 and 5).
Apoptotic bodies arising in tissues are quickly ingested by nearby cells and degraded within their lysosomes (Fig. 6 ) .There is no associated inflammation with
the outpouring of specialized phagocytes into the tissue, such as occurs with necrosis, and various types of
resident cells, including epithelial cells (Fig. 6 ) , participate in the mopping-up process. In tumors, viable neo-

Figure 5. Extensive apoptosis in human BM 13674 Burkitt's


lymphoma cell line 4 hours after heating to 43OC for 30 minutes
(electron micrograph, original magnification X4,600).

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Figure 6. Partly degraded apoptotic bodies containing recognizable


nuclear fragments within lysosomes in epithelial cell of murine small
intestinal crypt 2 hours after injection of cytosine arabinoside, 250
mg/kg (electron micrograph, original magnification X19,600).

plastic cells usually are involved, as are resident macrophages. However, apoptotic bodies formed in cell cultures mostly escape phagocytosis and eventually
degenerate.
The early cellular events in apoptosis are accomplished quickly, with only a few minutes elapsing between onset of the process and the formation of a cluster of apoptotic bodies. Thus, budding cells with convoluted outlines are rarely observed in tissue sections. The
small size of most apoptotic bodies makes them relatively inconspicuous by light microscopic study (Fig. 7).
After phagocytosis, their digestion is completed within
h o ~ r s . This
~ ~ ,fact
~ ~should be borne in mind when
apoptosis is being quantified histologically.
The distinction between apoptosis and necrosis is
unequivocal at the level of electron microscopic study
(Fig. l), and with practice, the two processes can be
recognized with confidence using light microscopic
study alone. Condensation of nuclear chromatin occurs
in the early stages of necrosis, but the chromatin is not
radically redistributed, as it is in apoptosis, and the
edges of the chromatin clumps tend to be irregular and
poorly defined (Fig. 8). In addition, the nucleus of the
necrotic cell never separates into discrete, membraneenclosed fragments. Late in necrosis, the chromatin disappears. The cytoplasm of the necrotic cell becomes
grossly swollen, and plasma and organelle membranes
progressively disintegrate (Fig. 8). Despite this, the
overall configuration of the cell tends to be preserved
until it is removed by mononuclear phagocytes. The

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Figure 7. Spontaneous apoptosis (arrows) occurring in poorly


differentiated human carcinoma (H & E, original magnification
X480).

involvement of groups of contiguous cells and the presence of an inflammatory exudate usually provide additional confirmatory evidence of categorization of the
cell death present in a particular circumstance as necrosis, In tumors, such foci of confluent necrosis typically
tend to be located in the centers of nodules, whereas
individual cells undergoing apoptosis are observed
throughout the viable tumor tissue (Fig. 7).

Figure 8. Spontaneous necrosis occurring in center of murine P-815


mastocytoma growing in muscle. Note the irregular clumping of
chromatin and disintegration of organelles and plasma membrane
(electron micrograph, original magnification X23,lOO).

Figure 9. Agarose gel electrophoresis of DNA extracted from


cultures of P-815 cells. Ethidium bromide stain photographed in
ultraviolet light. Lane 1: DRIgest 111molecular weight markers; lane
2: control culture; lane 3: culture showing extensive apoptosis
induced by heating; lane 4: culture showing massive necrosis 72
hours after repeated freezing and thawing.

Biochemical Mechanisms Involved in Execution


of Apoptosis

After apoptosis was defined as a morphologically distinct entity,5 some years elapsed before significant progress was made in elucidating its biochemical mechanism. In 1980, Wyllie4' showed that glucocorticoid-induced death of thymocytes, which was known to
display the typical ultrastructural features of apoptosis,
is associated with a unique change in the nuclear DNA.
There is double-strand cleavage at the linker regions
between nucleosomes, leading to the formation of fragments that are multiples of units comprising 180-200
base pairs. These fragments are detected readily by agarose gel electrophoresis, a characteristic ladder being
evident when ethidium bromide-stained gels are
viewed in ultraviolet light. Figure 9 shows such a ladder
produced by electrophoresis of DNA extracted from
apoptotic tumor cells. However, in necrosis there
usually is random cleavage of DNA and degradation of
h i ~ t o n e , ~a ~diffuse
* * ~ smear developing on DNA electrophoresis (Fig. 9). Internucleosomal cleavage has been
shown to accompany apoptosis occurring in a wide variety of cell types,43,56-59
and DNA electrophoresis is used
extensively for identifying the process. The cleavage
occasionally may be delayed or absent in cell death that
appears by other criteria to be apoptotic.60-62
It has been
found that internucleosomal cleavage is preceded by
cleavage of DNA into 300- or 50-kilo base-pair frag-

Apoptosis in Cancer/Kerr et al.

ments in cells undergoing apoptosis and that the formation of these large DNA fragments occurs in at least
some cases in which there is no subsequent development of oligonucleosomes.63The identity of the enzyme
or enzymes responsible for the internucleosomal cleavage is the subject of considerable debate.64-70Mitochondrial DNA does not appear to be cleaved.71r72
It has
been proposed that the cleavage of nuclear DNA at an
early stage of the process may serve a protective function in preventing the transfer of potentially active genetic material to nearby cells when apoptotic bodies are
phagocytosed.66
The cytoplasmic condensation, which is such a
prominent ultrastructural feature of apoptosis, is accompanied by an increase in cellular density." However,
nothing is known about the mechanisms involved. The
formation of the cell surface protuberances observed by
electron microscopic study has been shown by phasecontrast microscopic study to be associated with violent
convulsion of the cell ~ u r f a c e , and
~ ~ - this,
~ ~ taken in
conjunction with the subsequent separation of portions
of the cell to form membrane-bounded apoptotic bodies, clearly suggests major participation of cytoskeletal
elements. In cells destined to undergo apoptosis, p-tubulin messenger RNA increases before the development of morphologic changes and the occurrence of
DNA cleavage.77At a later stage, increased amounts of
0-tubulin appear in the cytoplasm. The P-tubulin genes
eventually are degraded along with the rest of the nuclear DNA once endonuclease becomes active.77Agents
that interfere with actin polymerization, such as cytochalasin B, have been shown to prevent the cellular
budding that leads to the formation of apoptotic bodies
without blocking fragmentation of the nucleus or DNA
cleavage.78
In the phase-contrast microscopic studies, the separation of discrete apoptotic bodies from the condensing
cell was found to coincide with abrupt cessation of cell
surface movement. A probable explanation for this observation has been provided by Fesus et al.49They have
shown that tissue-type transglutaminase, an enzyme
involved in the cross-linking of intracellular proteins, is
increased in cells undergoing a p o p t ~ s i s ~and
~ - ~that
' the
highest concentrations of the enzyme are consistently
present in discrete apoptotic bodies.83 They propose
that the transglutaminase activity leads to the formation of a highly cross-linked, rigid framework within
apoptotic bodies, which aids in maintaining their integrity and thus in preventing leakage of their contents
into the extracellular space. In several types of cells, the
increase in tissue transglutaminase activity associated
with apoptosis has been shown to be preceded by an
increase in the level of the corresponding messenger
RNA.4934

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The rapid phagocytosis of apoptotic bodies by


nearby cells while their membranes are intact implies
the operation of a highly specific recognition mechanism, and there is evidence that more than one such
mechanism may exist.85Early experiments showed that
the in vitro binding of apoptotic rodent thymocytes by
isologous peritoneal macrophages could be inhibited by
addition of N-acetyl glucosamine or its dimer N,N'diacetyl chitobiose,86and it was suggested that lectinlike receptors on the surface of the macrophages might
specifically recognize changes in the carbohydrates exposed on the surface of the apoptotic bodies. More recently, macrophage vitronectin receptors have been implicated in the recognition of neutrophil leukocytes undergoing a p o p t o s i ~ , ~and
~ , ~ ~evidence has been
produced that the exposure of phosphatidylserine on
the surface of apoptotic thymocytes and lymphocytes
may lead to their specific recognition by macrophagesE9The rapid phagocytosis of apoptotic bodies
before they lyse is of critical importance in preventing
inflammation and injury in the tissues in which they are
formed, especially when the process is occurring under
physiologic
Thus, there would have
been evolutionary advantage in the development of
multiple, and perhaps overlapping, mechanisms to ensure their immediate recognition by adjacent cells.85
Expression of several genes, in addition to those
mentioned, has been associated with the occurrence of
apoptosis, but whether their protein products are directly involved in initiation or execution of the process
remains unknown. The one most extensively studied is
TRPM-2. Its expression has been shown to be markedly
increased in a number of rodent tissues in which apoptosis is enhanced." However, the association does not
appear to be invariable.84z91
Recently, additional putative apoptosis-related genes have been identified using
subtractive hybridization technique^.^*-^^ Elucidation
of the functions of their protein products is awaited.
The occurrence of apoptosis in a number of circumstances has been shown to be suppressed by inhibitors
of messenger RNA or protein synthesis, such as actinomycin D and cycloheximide, r e s p e c t i ~ e l y . ~HOW~,~~-~~
ever, in other situations these inhibitors have no blocking effect; specific examples include apoptosis of target
cells induced by cytotoxic T - l y m p h ~ c y t e sapoptosis
,~~
of macrophages induced by g l i ~ t o x i and
n ~ ~apoptosis in
tumor cell lines induced by mild hyperthermia." In addition, to compound the problem, actinomycin D and
cycloheximide have been shown to induce apoptosis in
some normal and neoplastic cell populations.9~'00-'02
The significance of these conflicting findings is uncertain.
Theoretically, newly synthesized proteins might be
required for initiation of apoptosis by certain stimuli,

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for execution of the process, or for both. In the case of


and in rapidly proliferating populations, such as the
some triggering stimuli, there is evidence that cyclohexepithelium lining intestinal crypts'l5 and differentiating
imide exerts its blocking effect at the level of initiaspermatogonia."6 Although much of the cell loss in
tion,'03 and it can be argued that, when cycloheximide
populations of the latter type clearly is the result of
has no blocking effect, initiation occurs downstream in
shedding of cells from the tissue, in the former, mitosis
the activation pathway, bypassing the steps with a proand apoptosis balance each other under steady-state
tein synthetic requirement. However, evidence has
conditions. There is growing evidence that apoptosis is
been presented in the preceding paragraphs that proregulated in a reciprocal fashion to mitosis by growth
tein synthesis is needed for a number of the processes
factors and trophic hormones,"3~"4~1'7-'zz
and Raff' has
involved in the execution of apoptosis. Cohen and colsuggested that most cells in higher animals may require
l e a g u e ~have
~ ~ , suggested
~~
that, when apoptosis procontinuous trophic stimulation to survive. Raff postuceeds in the face of marked inhibition of protein synthelates that an increase in cell numbers in a particular
sis, the cells must possess all of the machinery necessary
location might lead to greater cellular competition for
for execution of the death sentence. Cohen also sugthe trophic factors that stimulate mitosis and inhibit
gests that, in cells in which protein synthesis inhibition
apoptosis and that this, in turn, might temporarily tip
activates apoptosis, the process normally is held in
the balance between the two processes, leading to restocheck by blocking proteins that have a short life span;
ration of the cell population to its former level. Howhe calls activation in these circumstances "the release
ever, there is evidence that substances that actively trigmechanism." However, the situation may be more
ger apoptosis also may be involved in normal cell popucomplex. This is suggested by the finding that, in some
lation homeostasis. In primary cultures of rabbit
cell populations, cycloheximide partially inhibits apopendometrial cells, factors that induce mitosis and apoptosis induced by certain stimuli, but cycloheximide intosis, respectively, have been found to be secreted in a
creases apoptosis above baseline levels in the same popcyclic but reciprocal fashion, with the result that cell
ulations when the agent is administered a l ~ n e . ' ~ ~ , ' ~numbers
~
show fluctuation on a daily basis but remain
Finally, there is a possible role for elevation of cytorelatively constant for extended periods of time.'23
solic Ca2+in triggering apoptosis. There is good eviA number of involutional processes occurring in
dence for such a mechanism in some cases,'06 but there
normal adult mammals have been shown to be assois equally compelling evidence that it is not involved in
ciated with marked enhancement of apoptosis; well~ t h e r s . ' ' ~The
~ ' ~matter
~
clearly is complex.'09
documented examples include reversion of the lactating
breast to its resting state after weaning,lZ4ovarian follicular a t r e ~ i a , ' ~and
~ ~ catagen
'~~
involution of hair folliIncidence of Apoptosis
c l e ~ . "The
~ trigger responsible for the increased apoptosis occurring during breast involution is likely to be
The circumstances of occurrence of apoptosis fall into
hormonal,'24 but in the other instances, the nature of
two broad categories. It accounts for the deletion of cells
that occurs in normal tissues, and it is observed in certhe initiating stimuli is uncertain.
In the immune system, apoptosis subserves special
tain specific pathologic contexts. In at least some of the
physiologic roles that are exclusive to the functional
latter, it can be argued teleologically that it subserves a
requirements of that system.48t5"For example, it is rebiologically meaningful, homeostatic function in deleting cells whose survival might be harmful to the
sponsible for the deletion of autoreactive T-cells in the
h ~ s t . ~ In
* , contrast,
~'
necrosis is always pathologic, bethymus that is responsible for self-tolerance'28 and for
ing the outcome of catastrophic injury to the cell.41No
selection of B-cells in lymphoid germinal centers during
humoral immune response^.'^^ Another specialized
homeostatic function can be attributed to it.
function of apoptosis in normal animals is the deletion
of effete cells, such as aging neutrophil leukocyte^'^'
Occurrence of Apoptosis in Normal Tissues
and megakaryocytes that have shed much of their cytoApoptosis plays an essential role in the normal developplasm during the formation of platelet^.'^'
ment of vertebrates. For example, it is responsible for
the regression of the tadpole tail that takes place during
Spontaneous Occurrence of Apoptosis in Tumors
metamorphosis into a frog"' and for removal of interdigital webs during limb development in mammalian
Apoptosis can be found in virtually all untreated maligembryos.53
nant t ~ m o r s , ' ~ and
~-'~
although
~
there have been few
In adult mammals, apoptosis occurs continually in
precise quantitative studies,'35histologic assessment inslowly proliferating cell populations, such as the epithedicates that its extent in some human tumors aplium of
p r ~ s t a t e , " and
~ adrenal ~ o r t e x , " ~ proaches that seen in rapidly involuting tissue^,^ indi-

Apoptosis in Cancer/Kerr et al.

cating that its kinetic significance must sometimes be


considerable.
The factors responsible for the spontaneous occurrence of apoptosis in tumors undoubtedly are diverse.
Apoptosis often is particularly prominent near foci of
confluent necrosis, where mild ischemia is likely to be
involved in its initiation; this is a known cause of enhancement of apoptosis in non-neoplastic tissues."'"36
Tumor necrosis factor a has been shown to induce
apoptosis in tumor cell lines in ~ i t r o , ' ~so
~ ,some
' ~ ~ of
the apoptosis observed in tumors in vivo may be attributable to release of this cytokine by infiltrating macrophages. In other instances, apoptosis may be a result of
attack on the tumor by cytotoxic T- lymphocyte^.'^'
However, increased apoptosis also is observed in preneoplastic foci and nodules developing in the liver after
administration of chemical carcinogen^^^"^^^^; it is unlikely that the factors mentioned would be operative in
these circumstances. It is possible that the putative cell
population regulatory mechanisms described earlier
come into play at an early stage of the process of carcinogenesis, with increased apoptosis temporarily balancing any increased cell proliferation that occurs, and that
much of the apoptosis observed in established tumors is
a result of the operation of these mechanisms. Finally,
increased apoptosis in tumors may result from processes intrinsic to the tumor cells, with differing rates of
apoptosis being found in otherwise similar tumors expressing different oncogenes."

Induction of Apoptosis by Radiation


Ionizing radiation, when given in small to moderate
doses, greatly enhances apoptosis in certain normal tissues without producing necrosis. Cells in the stem cell
region of hierarchically arranged rapidly proliferating
populations such as gut ~ r y p t s , ~ differentiating
,"~
spermatogonia,116rapidly proliferating cells in the fetus,I4'
and lymphocyte^^^^'^^^^^^ are particularly susceptible,
and it has been argued teleologically that the marked
propensity for such cells to undergo self-destruction
after the induction of DNA damage might reflect the
potential dangers associated with their persistence in
mutant form.51 Thus, persistence of stem cells with
unrepaired DNA damage would lead to immortalization of the genetic abn~rmalities"~;
one surviving mutant cell in a proliferating zone in the fetus would give
rise to many mutant progeny in the resulting mature
tissue; surviving mutant spermatogonia would give rise
to mutant gametes; and some lymphocytes with mutations in their receptor genes might have the capacity to
produce autoimmune disease.'45Of course, the occurrence of extensive apoptosis is damaging to the function
of a tissue. However, under natural conditions, animals

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encounter only small doses of radiation and deletion of


isolated cells with induced DNA damage in the tissues
listed would have afforded a selective advantage during evolution. Nevertheless, the differentiated acinar
cells of lacrimal and salivary glands have been found to
be susceptible to the induction of apoptosis by radiation, although bigger doses are r e q ~ i r e d . ' ~ ~This
- ' ~ ' susceptibility is not readily explicable on the basis of the
teleologic argument put forward.
There have been surprisingly few studies of apoptosis in irradiated tumors. However, it is clear that the
extent of apoptosis induced by radiation varies enormously from one tumor to a n ~ t h e r . ~ , Preliminary
~,~~',~~~
data suggest that there may be a correlation between
the magnitude of the immediate apoptotic response and
radiocurability,' but more studies are needed to examine this relationship.
The way in which radiation triggers the apoptotic
cascade in normal and neoplastic cells has been completely unknown until recently. It now seems possible
that the p53 tumor suppressor gene is involved (see
section on genetic regulation of apoptosis). It has been
suggested151that the product of the p53 gene acts as a
"molecular policeman," monitoring the integrity of the
genome. If DNA is damaged, the p53 product accumulates through a posttranslational stabilization mechanism and arrests the cell cycle at G1 to allow extra time
for repair. If repair fails, p53 may trigger deletion of the
cell by apoptosis. Cogent evidence for involvement of
the p53 gene in the induction of apoptosis by radiation
has been provided by the discovery that thymocytes
lacking p53 are resistant to the lethal effects of radiation
but retain their normal propensity to undergo apoptosis
after treatment with glucocorticoids.152~153
However, it
should be noted that the last step in the sequence proposed, induction of apoptosis by an increase in the level
of the normal (wild-type) p53 gene product, appears to
have been demonstrated only in tumor-derived cell
lines.28*36

Induction of Apoptosis by Cancer Chemotherapeutic


Agents
A variety of anti-cancer drugs have been shown to induce extensive apoptosis in rapidly proliferating normal cell populations, lymphoid tissues, and tumors.'15,116,154
Thus, enhanced apoptosis is responsible for
many of the adverse effects of chemotherapy and for
tumor regression.
The way in which anti-cancer drugs induce apoptosis is unknown.155Better understanding of the processes involved clearly might be expected to lead to improved treatment regimen^.'^' However, there is an additional important consequence of the realization that

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CANCER April 25, 1994, Volume 73,No. 8

anti-cancer drugs mediate their therapeutic effect by


triggering apoptosis. As has been stressed, apoptosis is
a regulated phenomenon capable of being inhibited
and activated. Herein may lie a novel explanation for
certain instances of drug resistance. Indeed, there is evidence that stimulation of some cell lines by trophic cytokines or increase in their level of expression of the
bcl-2 proto-oncogene (the bcl-2 gene product inhibits
apoptosis occurring in a variety of circumstances; see
section on genetic regulation) can greatly increase their
resistance to the apoptosis-inducing effect of anticancer d r u g ~ . ~ ~ , ' ~ ~ - ' ~ ~

sociated with resistance to induction of apoptosis by


glucocorticoids in several lymphoid cell lines.29r35

Induction of Apoptosis by Antibodies to the APO-1


or Fas Antigen

The APO-1 antigen was defined during studies of


monoclonal antibodies raised against a human B-lymphoblast cell
One of the antibodies was found to
induce apoptosis of activated human B- and T-lymphocytes and of the cells of a variety of human lymphoid
tumor-derived cell lines. The cell membrane antigen to
which this antibody attaches was designated APO-1 .164
Induction of Apoptosis by Mild Hyperthermia
The Fas antigen, defined by a second monoclonal antibody developed by another group of workers,'65 has
In susceptible tissues, heating to 43C for 30 minutes
been found to be identical to the APO-1 antigen.'66 The
induces extensive apoptosis, whereas heating to tempermolecule belongs to the human tumor necrosis factor
atures of 46OC and greater for similar periods produces
receptor/nerve growth factor receptor superfamily of
necrosis.'6 The spectrum of tissue susceptibility to
cell surface protein^.'^^,'^^
apoptosis induction by hyperthermia is essentially simiInjection of anti-APO-1 monoclonal antibodies
lar to that described previously for radiation and anticauses rapid regression of murine xenografts of APOcancer drugs-rapidly proliferating normal cell populal-expressing human lymphoid cell lines, with the retions, lymphoid organs, and tumors.'0~"~'6~'8~'59~'61
As is
gression being accompanied by greatly enhanced apopthe case with the other two agents, there is considerable
tosis of the grafted cell^.'^^,'^^ It is not known whether
variability in the response of tumors from one to anthe effect of anti-APO-1 antibodies on normal cells
other.17No definitive information is available on how
would preclude their administration to humans. Howhyperthermia induces apoptosis. Its full potential as a
ever, additional study of this receptor and a search for
therapeutic agent for cancer probably will not be realother similar receptors may yield results with therapeuized until its mechanism of action is better understood.
tic applications."j8

Induction of Apoptosis by Hormone Withdrawal or


Addition
Apoptosis is involved in the atrophy of endocrine-dependent organs, such as the p r ~ s t a t e ~ ~
and
, " ~adrenal
~ortex,"~
that follows withdrawal of trophic hormonal
stimulation, and as might be expected, it also is enhanced in hormone-dependent tumors after successful
ablation the rap^.'^-'^ In contrast, increased levels of
glucocorticoid induce apoptosis of thym~cytes,~'
and a
similar effect is observed with many lymphocytic leukemias and malignant lymphomas.'02,'62
In view of the possible role of increased bcl-2
proto-oncogene expression in the development of resistance of tumors to anti-cancer drugs, it is of great interest that recent reports indicate that it also may be involved in resistance to hormone therapy. Thus, although bcl-2 expression was found to be virtually
undetectable by immunohistochemistry in 13 of 19
cases of androgen-dependent human prostatic cancer,
all of the androgen-independent cancers studied, with
the exception of tissue obtained from bone marrow metastases, displayed positive staining for bcl-2 protein.'63
In addition, bci-2 expression has been shown to be as-

Induction of Apoptosis by Cytotoxic Lymphocytes


We conclude our survey of the incidence of apoptosis
with a brief reference to its involvement in cell-mediated immune killing.
In vitro studies have shown that target cell death
induced by T-cells,43,75,76,169,170
K-cells171and NK cells'72
is apoptotic in type, and enhanced apoptosis has been
observed in vivo in cellular immune rejection of allog r a f t ~ and
' ~ ~in graft-versus-host disease.53Deletion of
virus-infected cells by cytotoxic lymphocytes plays an
essential role in the elimination of viruses from the
body, and involvement of apoptosis in this deletion
clearly exemplifies its homeostatic function.'74 Apoptosis induced by cytotoxic T-cells is not blocked by inhibitors of protein synthesis43or by bcl-2 e x p r e ~ s i o nDis.~~
tinctive activation mechanisms probably are involved. 175
Genetic Regulation of Apoptosis
In this article we have referred to the regulation of
apoptosis by certain proto-oncogenes and the p53 tu-

Apoptosis in Cancer/Kerr et al.

mor suppressor gene. We now briefly review research


in this area in a more systemic fashion.

Znvolvement of the c-myc and c-fos Proto-oncogenes


in the Induction of Apoptosis

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myeloid and lymphoid cell lines promoted survival of


these cells after withdrawal of interleukin-3, but did not
stimulate their pr~liferation.~
Subsequently, the gene
was shown to specifically inhibit apopt~sis.~
Thus, bcl2 emerged as a new type of proto-oncogene, one that
suppresses cell death rather than stimulating proliferation. However, it does not inhibit apoptosis occurring in
all circumstances; as has been mentioned, it fails to
block apoptosis induced by cytotoxic T-lymphocyte~.~~
The topographic distribution of bcl-2 expression in
normal tissues suggests that it plays vital roles in a variety of physiologic processes in which differential cell
survival is imp~rtant.~
As far as oncogenesis is concerned, in addition to the synergy with c-myc mentioned, deregulation of bcl-2 expression may contribute
to the accumulation of oncogenic mutations by suppressing the apoptotic deletion of cells that normally
follows the induction of DNA damage by agents such
as r a d i a t i ~ n . ~ ~

In 1988, Buttyan et aLZ3recorded a marked increase in


the amount of c-myc messenger RNA in the rat ventral
prostate gland after castration, with peak levels of the
transcript occurring at the stage of involution when
apoptosis is at its maximum. The c-fos gene also was
found to be induced, but at an earlier time than was
c-myc. These authors concluded that mitosis and apoptosis might share common signal pathways.
More recently it has been shown that antisense oligonucleotides corresponding to c-myc block activation-induced apoptosis in T-cell h y b r i d ~ m a sa, result
~~
that suggests that c-myc expression might be required
for the initiation of apoptosis. That such a requirement
is not universal is shown by the antisense oligonucleotides having no effect on the induction of apoptosis in
Enhancement of Apoptosis After Znduced Expression
the same hybridomas by glucocorticoids.37
of the p53 Tumor Suppressor Gene
The paradoxical involvement of c-myc in the regulation of mitosis and apoptosis has been clarified, to some
Abnormalities of the p53 tumor suppressor gene, rangextent, by experiments on cell lines that overexpress
ing from complete deletion to point mutation, constic-myc as a consequence of gene t r a n ~ f e r . ~ ~ Al, ~ ~ , ~tute
, ~ some
~
of the most frequently encountered genetic
though the cells of such lines continue to proliferate in
defects in human cancer.177This clearly suggests that
media containing high concentrations of serum, they
p53 plays a central role in the regulation of cell proliferaexhibit extensive apoptosis when grown in low serum
tion. By introducing the wild-type p53 gene into cell
media30,32,33.
, in contrast, normal cells downregulate clines lacking normal p53 activity, a number of p53-memyc under these circumstances and pass into a quiesdiated functions have been identified.77These include
cent state.32In addition, the lines that overexpress cgrowth arrest, which occurs primarily in the G I phase
myc exhibit accelerated apoptosis on withdrawal of
of the cell cycle, and cellular differentiation. However,
growth
Thus, increased c-myc expression
two p53-negative cell lines, derived from a mouse with
can result in mitosis or apoptosis, depending on the
myeloid leukemia and a human colon tumor, respecavailability of other critical growth stimuli.52In the prestively, have been found to respond to induced expresence of such stimuli, c-myc acts as a classic proto-oncosion of wild-type p53 with the extensive occurrence of
gene, stimulating mitosis; in their absence, it initiates
apoptosi~.~*~~
apoptosis. Simultaneous overexpression of the bcl-2
To what extent p53 is involved in regulating apopproto-oncogene abrogates the capacity of increased cis ~notetosis under normal conditions is ~ n k n 0 w n .Itl ~
myc expression to induce apoptosis, a fact that may be
worthy that p53-deficient mice develop normally but
of importance in the synergistic involvement of these
are susceptible to spontaneous turn or^.'^' As indicated,
two genes in o n c o g e n e ~ i s . ~ , ~ ~ , ~ ~
there is evidence that p53 is involved in triggering the
The possible involvement of c-fos expression in the
apoptotic deletion of cells that have sustained DNA
initiation of apoptosis has been critically reviewed.76
damage. A major mechanism whereby abnormalities of
p53 contribute to the development and progression of
tumors may be abrogation of the normal pathway that
Inhibition of Apoptosis by the bcl-2 Proto-oncogene
leads
to the self-destruction of mutant cells.79
Product
Bcl-2 originally was proposed as a candidate proto-oncogene because of its location at a breakpoint in a chromosome translocation that occurs in a proportion of
human B-cell lymphomas.34In 1988 it was shown that
introduction of the gene into interleukin-3-dependent

Conclusion

The primary importance of the apoptosis concept for


oncology lies in its being a regulated phenomenon subject to stimulation and inhibition. Although little is

2022

CANCER April 15, 1994, Volume 73,No. 8

known about how established therapeutic agents for


cancer effect its initiation, it seems reasonable to suggest that greater understanding of the processes involved might lead to the development of improved
treatment regimens. Inhibitory mechanisms such as
bcl-2 proto-oncogene expression may be implicated in
the development of resistance of tumors to therapeutic
agents, and may contribute to tumor growth and perhaps to oncogenesis by allowing the inappropriate survival of cells with DNA abnormalities. It is likely that
additional inhibitory mechanisms will be defined. Finally, the discovery that monoclonal antibodies can induce apoptosis of lymphoid tumor cells via the APO-1
or Fas receptor may have implications for the development of novel approaches to therapy. Additional receptors of this type should be sought.
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