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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 re- sponsible
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 bod- ies containing well-preserved
organelles, which are phagocytosed and digested by nearby resident
cells. There is no associated inflammation. A characteristic bio-
chemical feature of the process is double-strand cleavage of
nuclear DNA at the linker regions between nucleo- somes leading to
the production of oligonucleosomal fragments. In many, although not
all of the circum- stances in which apoptosis occurs, it is
suppressed by inhibitors of messenger RNA and protein synthesis.
Apoptosis occurs spontaneously in malignant tumors, of- ten
markedly retarding their growth, and it is increased in tumors
responding to irradiation, cytotoxic chemother- apy, heating and
hormone ablation. However, much of the current interest in the
process stems from the discov- ery that it can be regulated by
certain proto-oncogenes and the p53 tumor suppressor gene. Thus,
c-myc expres- sion 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 tumor- derived cell lines
transfected with wild-type p53, induc- tion 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 pro- tein 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, Queens-
land, Australia.
Supported by the Queensland Cancer Fund and the University of
Queensland.
Address for reprints: John F. R. Kerr, Ph.D., Department of Pa-
thology, University of Queensland Medical School, Herston, Bris-
bane, Queensland 4006, Australia.
Accepted for publication November 24, 1993.
Key words: apoptosis, programmed cell death, cell dele- tion,
DNA fragmentation, radiation, anti-cancer drugs, hyperthermia,
hormone ablation, proto-oncogene, tumor suppressor gene.
Oncologists traditionally have been concerned primar- ily with
cell proliferation. However, apoptosis (the dis- tinctive form of
cell death that complements cell prolif- eration 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-'8 and hormone abla-
tion.l9-'' However, during the past few years, advances in
understanding of the control of apoptosis at the mo- lecular level
have extended its potential oncologic signif - 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 prod- ucts of certain proto-oncogenes and the p53
tumor sup- pressor has opened up exciting avenues for future
re~earch.~
The proposition that apoptosis is a discrete phenom- enon that
is fundamentally different from degenerative cell death or necrosis
is based on its morphology, bio- chemistry, 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 apopto- tic bodies
(Fig. 5). The size and composition of the latter vary considerably;
many contain several nuclear frag- ments (Fig. 5) whereas others
lack a nuclear compo- nent. In addition, the extent of the nuclear
and cellular budding vanes with cell type, often being relatively
re- stricted in small cells with a high nucleocytoplasmic
statement with lists of references but will give a compos- ite
overview; supporting bibliographies and many ad- ditional
illustrations can be found in comprehensive re- views.41.53,54
The contrasting ultrastructural features of apopto- sis 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 chroma- tin, with the formation of
sharply delineated, uniformly finely granular masses that become
marginated against the nuclear envelope, and condensation of the
cyto- plasm. Progression of the condensation is accompanied by
convo~u~on of the nuclear and cell outlines, and this is into
discrete fragments that are surrounded by a double-layered en-
velope (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).
by breaking up Of the
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Apoptosis in Cancer/Kerr et al . 2015
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 pre- served (Figs. 4 and
5).
Apoptotic bodies arising in tissues are quickly in- gested by
nearby cells and degraded within their lyso- somes (Fig. 6) . There
is no associated inflammation with the outpouring of specialized
phagocytes into the tis- sue, such as occurs with necrosis, and
various types of resident cells, including epithelial cells (Fig.
6) , partici- pate 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).
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 macro-
phages. However, apoptotic bodies formed in cell cul- tures mostly
escape phagocytosis and eventually degenerate.
The early cellular events in apoptosis are accom- plished
quickly, with only a few minutes elapsing be- tween onset of the
process and the formation of a clus- ter of apoptotic bodies. Thus,
budding cells with convo- luted outlines are rarely observed in
tissue sections. The small size of most apoptotic bodies makes them
rela- tively 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, membrane- enclosed fragments. Late in necrosis, the
chromatin dis- appears. 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 pres- ence of
an inflammatory exudate usually provide addi- tional confirmatory
evidence of categorization of the cell death present in a
particular circumstance as necro- sis, 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 111 molecular 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 dis- tinct
entity,5 some years elapsed before significant pro- gress was made
in elucidating its biochemical mecha- nism. In 1980, Wyllie4'
showed that glucocorticoid-in- duced 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 frag- ments that are multiples of units
comprising 180-200 base pairs. These fragments are detected readily
by aga- rose 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 electro- phoresis (Fig. 9). Internucleosomal cleavage has
been shown to accompany apoptosis occurring in a wide vari- ety 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-
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Apoptosis in Cancer/Kerr et al . 2017
ments in cells undergoing apoptosis and that the for- mation of
these large DNA fragments occurs in at least some cases in which
there is no subsequent develop- ment of oligonucleosomes.63 The
identity of the enzyme or enzymes responsible for the
internucleosomal cleav- age is the subject of considerable
debate.64-70 Mito- chondrial 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 func- tion
in preventing the transfer of potentially active ge- netic material
to nearby cells when apoptotic bodies are phagocytosed.66
The cytoplasmic condensation, which is such a prominent
ultrastructural feature of apoptosis, is accom- panied 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 phase- contrast 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 bod- ies, clearly suggests major
participation of cytoskeletal elements. In cells destined to
undergo apoptosis, p-tu- bulin messenger RNA increases before the
develop- ment of morphologic changes and the occurrence of DNA
cleavage.77 At 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 nu- clear DNA once endonuclease becomes
active.77 Agents that interfere with actin polymerization, such as
cyto- chalasin 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 sepa- ration of
discrete apoptotic bodies from the condensing cell was found to
coincide with abrupt cessation of cell surface movement. A probable
explanation for this ob- servation has been provided by Fesus et
al.49 They 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 forma- tion of a highly
cross-linked, rigid framework within apoptotic bodies, which aids
in maintaining their integ- rity 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
The rapid phagocytosis of apoptotic bodies by nearby cells while
their membranes are intact implies the operation of a highly
specific recognition mecha- nism, and there is evidence that more
than one such mechanism may exist.85 Early 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,86 and it was
suggested that lectin- like receptors on the surface of the
macrophages might specifically recognize changes in the
carbohydrates ex- posed on the surface of the apoptotic bodies.
More re- cently, macrophage vitronectin receptors have been im-
plicated in the recognition of neutrophil leukocytes un- dergoing 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 macro- phagesE9 The 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 en- sure 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 di- rectly 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 apop- tosis is enhanced."
However, the association does not appear to be invariable.84z91
Recently, additional puta- tive 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 circum- stances has
been shown to be suppressed by inhibitors of messenger RNA or
protein synthesis, such as actino-
ever, in other situations these inhibitors have no block- ing
effect; specific examples include apoptosis of target cells induced
by cytotoxic T-lymph~cytes ,~~ apoptosis of macrophages induced by
g l i ~ t o x i n ~ ~ and apoptosis in tumor cell lines induced by
mild hyperthermia." In ad- dition, 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 uncer- tain.
Theoretically, newly synthesized proteins might be required for
initiation of apoptosis by certain stimuli,
mycin D and cycloheximide, r e s p e c t i ~ e l y . ~ ~ , ~ ~ -
~ ~ HOW-
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2018 CANCER April 25, 1994, Volume 73, No. 8
for execution of the process, or for both. In the case of some
triggering stimuli, there is evidence that cyclohex- imide exerts
its blocking effect at the level of initia- tion,'03 and it can be
argued that, when cycloheximide has no blocking effect, initiation
occurs downstream in the activation pathway, bypassing the steps
with a pro- tein synthetic requirement. However, evidence has been
presented in the preceding paragraphs that pro- tein synthesis is
needed for a number of the processes involved in the execution of
apoptosis. Cohen and col- l e a g u e ~ ~ ~ , ~ ~ have suggested
that, when apoptosis pro- ceeds in the face of marked inhibition of
protein synthe- sis, the cells must possess all of the machinery
necessary for execution of the death sentence. Cohen also sug-
gests that, in cells in which protein synthesis inhibition
activates apoptosis, the process normally is held in check by
blocking proteins that have a short life span; he calls activation
in these circumstances "the release mechanism." However, the
situation may be more complex. This is suggested by the finding
that, in some cell populations, cycloheximide partially inhibits
apop- tosis induced by certain stimuli, but cycloheximide in-
creases apoptosis above baseline levels in the same pop- ulations
when the agent is administered a l ~ n e . ' ~ ~ , ' ~ ~
Finally, there is a possible role for elevation of cyto- solic
Ca2+ in triggering apoptosis. There is good evi- dence for such a
mechanism in some cases,'06 but there is equally compelling
evidence that it is not involved in ~ t h e r s . ' ' ~ ~ ' ~ ~ The
matter clearly is complex.'09
Incidence of Apoptosis
The circumstances of occurrence of apoptosis fall into two broad
categories. It accounts for the deletion of cells that occurs in
normal tissues, and it is observed in cer- tain specific pathologic
contexts. In at least some of the latter, it can be argued
teleologically that it subserves a biologically meaningful,
homeostatic function in delet- ing cells whose survival might be
harmful to the h ~ s t . ~ * , ~ ' In contrast, necrosis is always
pathologic, be- ing the outcome of catastrophic injury to the
cell.41 No homeostatic function can be attributed to it.
Occurrence of Apoptosis in Normal Tissues
Apoptosis plays an essential role in the normal develop- ment of
vertebrates. For example, it is responsible for the regression of
the tadpole tail that takes place during metamorphosis into a
frog"' and for removal of inter- digital webs during limb
development in mammalian embryos.53
In adult mammals, apoptosis occurs continually in slowly
proliferating cell populations, such as the epithe- lium of
pr~state ,"~ and adrenal ~ortex,"~
and in rapidly proliferating populations, such as the epithelium
lining intestinal crypts'l5 and differentiating spermatogonia."6
Although much of the cell loss in populations of the latter type
clearly is the result of shedding of cells from the tissue, in the
former, mitosis and apoptosis balance each other under steady-state
conditions. There is growing evidence that apoptosis is regulated
in a reciprocal fashion to mitosis by growth factors and trophic
hormones,"3~"4~1'7-'zz and Raff' has suggested that most cells in
higher animals may require continuous trophic stimulation to
survive. Raff postu- lates that an increase in cell numbers in a
particular location might lead to greater cellular competition for
the trophic factors that stimulate mitosis and inhibit apoptosis
and that this, in turn, might temporarily tip the balance between
the two processes, leading to resto- ration of the cell population
to its former level. How- ever, there is evidence that substances
that actively trig- ger apoptosis also may be involved in normal
cell popu- lation homeostasis. In primary cultures of rabbit
endometrial cells, factors that induce mitosis and apop- tosis,
respectively, have been found to be secreted in a cyclic but
reciprocal fashion, with the result that cell numbers show
fluctuation on a daily basis but remain relatively constant for
extended periods of time.'23
A number of involutional processes occurring in normal adult
mammals have been shown to be asso- ciated with marked enhancement
of apoptosis; well- documented examples include reversion of the
lactating breast to its resting state after weaning,lZ4 ovarian
follic- ular a t r e ~ i a , ' ~ ~ ~ ' ~ ~ and catagen involution
of hair folli- c l e ~ . " ~ The trigger responsible for the
increased apop- tosis occurring during breast involution is likely
to be hormonal,'24 but in the other instances, the nature of the
initiating stimuli is uncertain.
In the immune system, apoptosis subserves special physiologic
roles that are exclusive to the functional requirements of that
system.48t5" For example, it is re- sponsible for the deletion of
autoreactive T-cells in the thymus that is responsible for
self-tolerance'28 and for selection of B-cells in lymphoid germinal
centers during humoral immune response^.'^^ Another specialized
function of apoptosis in normal animals is the deletion of effete
cells, such as aging neutrophil leukocyte^'^' and megakaryocytes
that have shed much of their cyto- plasm during the formation of
platelet^.'^'
Spontaneous Occurrence of Apoptosis in Tumors
Apoptosis can be found in virtually all untreated malig- nant t
~ m o r s , ' ~ ~ - ' ~ ~ and although there have been few precise
quantitative studies,'35 histologic assessment in- dicates that its
extent in some human tumors ap- proaches that seen in rapidly
involuting tissue^,^ indi-
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Apoptosis in Cancer/Kerr et al . 2019
cating that its kinetic significance must sometimes be
considerable.
The factors responsible for the spontaneous occur- rence 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 en- hancement 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 attrib- utable
to release of this cytokine by infiltrating macro- phages. In other
instances, apoptosis may be a result of attack on the tumor by
cytotoxic T- lymphocyte^.'^' However, increased apoptosis also is
observed in pre- neoplastic foci and nodules developing in the
liver after administration of chemical carcinogen^^^"^^^^; it is
un- likely 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 carci- nogenesis, with increased apoptosis
temporarily balanc- ing 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 pro- cesses intrinsic
to the tumor cells, with differing rates of apoptosis being found
in otherwise similar tumors ex- pressing different oncogenes."
Induction of Apoptosis by Radiation
Ionizing radiation, when given in small to moderate doses,
greatly enhances apoptosis in certain normal tis- sues without
producing necrosis. Cells in the stem cell region of hierarchically
arranged rapidly proliferating populations such as gut ~ r y p t s
, ~ , " ~ differentiating sper- matogonia,116 rapidly 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
immortaliza- tion of the genetic abn~rmalities"~; one surviving mu-
tant 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 muta- tions in their receptor genes might have the
capacity to produce autoimmune disease.'45 Of course, the occur-
rence of extensive apoptosis is damaging to the function of a
tissue. However, under natural conditions, animals
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 dur- ing evolution. Nevertheless,
the differentiated acinar cells of lacrimal and salivary glands
have been found to be susceptible to the induction of apoptosis by
radia- tion, although bigger doses are req~ired. '~~- '~ ' This
sus- ceptibility is not readily explicable on the basis of the
teleologic argument put forward.
There have been surprisingly few studies of apop- tosis in
irradiated tumors. However, it is clear that the extent of
apoptosis induced by radiation varies enor- mously 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 exam- ine this relationship.
The way in which radiation triggers the apoptotic cascade in
normal and neoplastic cells has been com- pletely 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 suggested151 that the product of the p53 gene acts as a
"molecular policeman," monitoring the integrity of the genome. If
DNA is damaged, the p53 product accumu- lates through a
posttranslational stabilization mecha- nism 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 pro- posed, 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 in- duce
extensive apoptosis in rapidly proliferating nor- mal cell
populations, lymphoid tissues, and tumors.'-
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 apop- tosis is
unknown.155 Better understanding of the pro- cesses involved
clearly might be expected to lead to im- proved treatment
regimen^.'^' However, there is an ad- ditional important
consequence of the realization that
15,116,154
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2020 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 evi- dence that stimulation of some cell lines by
trophic cy- tokines 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 anti- cancer d r u g ~ . ~ ~ , ' ~ ~ -
' ~ ~
Induction of Apoptosis by Mild Hyperthermia
In susceptible tissues, heating to 43C for 30 minutes induces
extensive apoptosis, whereas heating to temper- atures of 46OC and
greater for similar periods produces necrosis.'6 The spectrum of
tissue susceptibility to apoptosis induction by hyperthermia is
essentially simi- lar to that described previously for radiation
and anti- cancer drugs-rapidly proliferating normal cell popula-
tions, lymphoid organs, and tumors.'0~"~'6~'8~'59~'61 As is the
case with the other two agents, there is considerable variability
in the response of tumors from one to an- other.17 No definitive
information is available on how hyperthermia induces apoptosis. Its
full potential as a therapeutic agent for cancer probably will not
be real- ized until its mechanism of action is better
understood.
Induction of Apoptosis by Hormone Withdrawal or Addition
Apoptosis is involved in the atrophy of endocrine-de- pendent
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 en- hanced 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 leu- kemias and malignant lymphomas.'02,'62
In view of the possible role of increased bcl-2 proto-oncogene
expression in the development of resis- tance of tumors to
anti-cancer drugs, it is of great inter- est that recent reports
indicate that it also may be in- volved in resistance to hormone
therapy. Thus, al- though 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 me- tastases, displayed positive staining
for bcl-2 protein.'63 In addition, bci-2 expression has been shown
to be as-
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-lym- phoblast cell One of the
antibodies was found to induce apoptosis of activated human B- and
T-lympho- cytes 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 The Fas antigen,
defined by a second monoclonal anti- body developed by another
group of workers,'65 has been found to be identical to the APO-1
antigen.'66 The molecule belongs to the human tumor necrosis factor
receptor/nerve growth factor receptor superfamily of cell surface
protein^.'^^,'^^
Injection of anti-APO-1 monoclonal antibodies causes rapid
regression of murine xenografts of APO- l-expressing human lymphoid
cell lines, with the re- gression being accompanied by greatly
enhanced apop- tosis of the grafted cell^.'^^,'^^ It is not known
whether the effect of anti-APO-1 antibodies on normal cells would
preclude their administration to humans. How- ever, additional
study of this receptor and a search for other similar receptors may
yield results with therapeu- tic applications."j8
Induction of Apoptosis by Cytotoxic Lymphocytes
We conclude our survey of the incidence of apoptosis with a
brief reference to its involvement in cell-me- diated immune
killing.
In vitro studies have shown that target cell death induced by
T-cells,43,75,76,169,170 K-cells171 and NK cells'72 is apoptotic in
type, and enhanced apoptosis has been observed in vivo in cellular
immune rejection of allo- g r a f t ~ ' ~ ~ and in
graft-versus-host disease.53 Deletion 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 Apopto-
sis induced by cytotoxic T-cells is not blocked by inhibi- tors of
protein synthesis43 or by bcl-2 expre~s ion .~~ Dis- tinctive
activation mechanisms probably are in- volved. 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. 2021
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
In 1988, Buttyan et aLZ3 recorded 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 apop- tosis might
share common signal pathways.
More recently it has been shown that antisense oli-
gonucleotides corresponding to c-myc block activa- tion-induced
apoptosis in T-cell h y b r i d ~ m a s , ~ ~ a 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 oligonucleo- tides having no effect on the induction
of apoptosis in the same hybridomas by glucocorticoids.37
The paradoxical involvement of c-myc in the regula- tion of
mitosis and apoptosis has been clarified, to some extent, by
experiments on cell lines that overexpress
though the cells of such lines continue to proliferate in media
containing high concentrations of serum, they exhibit extensive
apoptosis when grown in low serum
myc under these circumstances and pass into a quies- cent
state.32 In addition, the lines that overexpress c- myc exhibit
accelerated apoptosis on withdrawal of growth Thus, increased c-myc
expression can result in mitosis or apoptosis, depending on the
availability of other critical growth stimuli.52 In the pres- ence
of such stimuli, c-myc acts as a classic proto-onco- gene,
stimulating mitosis; in their absence, it initiates apoptosis.
Simultaneous overexpression of the bcl-2 proto-oncogene abrogates
the capacity of increased c- myc expression to induce apoptosis, a
fact that may be of importance in the synergistic involvement of
these two genes in oncogene~is . ~ ,~~,~~
The possible involvement of c-fos expression in the initiation
of apoptosis has been critically reviewed.76
c-myc as a consequence of gene t r a n ~ f e r . ~ ~ , ~ ~ , ~ ,
~ ~ Al-
media30,32,33. , in contrast, normal cells downregulate c-
Inhibition of Apoptosis by the bcl-2 Proto-oncogene Product
Bcl-2 originally was proposed as a candidate proto-on- cogene
because of its location at a breakpoint in a chro- mosome
translocation that occurs in a proportion of human B-cell
lymphomas.34 In 1988 it was shown that introduction of the gene
into interleukin-3-dependent
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, bcl- 2 emerged as a new type of
proto-oncogene, one that suppresses cell death rather than
stimulating prolifera- tion. 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 vari- ety of
physiologic processes in which differential cell survival is
imp~rtant.~ As far as oncogenesis is con- cerned, in addition to
the synergy with c-myc men- tioned, deregulation of bcl-2
expression may contribute to the accumulation of oncogenic
mutations by sup- pressing the apoptotic deletion of cells that
normally follows the induction of DNA damage by agents such as rad
ia t i~n . ~~
Enhancement of Apoptosis After Znduced Expression of the p53
Tumor Suppressor Gene
Abnormalities of the p53 tumor suppressor gene, rang- ing from
complete deletion to point mutation, consti- tute some of the most
frequently encountered genetic defects in human cancer.177 This
clearly suggests that p53 plays a central role in the regulation of
cell prolifera- tion. By introducing the wild-type p53 gene into
cell lines lacking normal p53 activity, a number of p53-me- diated
functions have been identified.77 These include growth arrest,
which occurs primarily in the GI phase of the cell cycle, and
cellular differentiation. However, two p53-negative cell lines,
derived from a mouse with myeloid leukemia and a human colon tumor,
respec- tively, have been found to respond to induced expres- sion
of wild-type p53 with the extensive occurrence of apoptos i~ .
~*~~
To what extent p53 is involved in regulating apop- tosis under
normal conditions is ~ n k n 0 w n . l ~ ~ It is note- worthy that
p53-deficient mice develop normally but are susceptible to
spontaneous turn or^.'^' As indicated, there is evidence that p53
is involved in triggering the apoptotic deletion of cells that have
sustained DNA damage. A major mechanism whereby abnormalities of
p53 contribute to the development and progression of tumors may be
abrogation of the normal pathway that leads to the self-destruction
of mutant cells.79
Conclusion
The primary importance of the apoptosis concept for oncology
lies in its being a regulated phenomenon sub- ject to stimulation
and inhibition. Although little is
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2022 CANCER April 15, 1994, Volume 73, No. 8
known about how established therapeutic agents for cancer effect
its initiation, it seems reasonable to sug- gest that greater
understanding of the processes in- volved 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 per- haps to oncogenesis by allowing
the inappropriate sur- vival of cells with DNA abnormalities. It is
likely that additional inhibitory mechanisms will be defined. Fi-
nally, the discovery that monoclonal antibodies can in- duce
apoptosis of lymphoid tumor cells via the APO-1 or Fas receptor may
have implications for the develop- ment of novel approaches to
therapy. Additional recep- tors of this type should be sought.
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