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-'8and 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 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 prod-
ucts of certain proto-oncogenes and the p53 tumor sup-
pressor has opened up exciting avenues for
future r e ~ e a r c h . ~
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
2014 CANCER April 15,1994, Volume 73,No. 8

~ 3

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

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.
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 comprehensivere-
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
ofnthe nuclear and cell outlines, and this
by c o n v o ~ u ~ o Figure 3. Same culture as that illustrated in Figure 2. Separation of
surface protuberances is leading to apoptotic body formation. Some
is by breaking up Of the into discrete nuclear fragments show peripheral masses of condensed chromatin,
fragments that are surrounded by a double-layered en- whereas others are uniformly dense in plane of section (electron
velope (Fig. 2) and by budding of the cell as a whole 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 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).
2015
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
2016 CANCER April 25, 2994, Volume 73, No. 8
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 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 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-
Apoptosis in Cancer/Kerr et al.
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.63The identity of the enzyme
or enzymes responsible for the internucleosomal cleav-
age is the subject of considerable debate.64-70Mito-
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.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 nu-
clear DNA once endonuclease becomes active.77Agents
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.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 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
2017
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.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 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-
phagesE9The 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-
mycin D and cycloheximide, r e s p e c t i ~ e l y . ~HOW-
~,~~-~~
ever, in other situations these inhibitors have no block-
ing 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 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,
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 . ' ~ ~ , ' ~numbers
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.41No
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 ~ adrenal ~ o r t e x , " ~ proaches that seen in rapidly involuting tissue^,^ indi-
p r ~ s t a t e , " and
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
~ 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,lZ4ovarian 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,'35histologic assessment in-
dicates that its extent in some human tumors ap-
Apoptosis in Cancer/Kerr et al.
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,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 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.'45Of course, the occur-
rence of extensive apoptosis is damaging to the function
of a tissue. However, under natural conditions, animals
2019
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 r e q ~ i r e d . ' ~ ~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
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 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.'-
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 apop-
tosis is unknown.155Better 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
2020 CANCER April 25, 1994, Volume 73,No. 8

anti-cancer drugs mediate their therapeutic effect by sociated with resistance to induction of apoptosis by
triggering apoptosis. As has been stressed, apoptosis is glucocorticoids in several lymphoid cell lines.29r35
a regulated phenomenon capable of being inhibited
and activated. Herein may lie a novel explanation for
Induction of Apoptosis by Antibodies to the APO-1
certain instances of drug resistance. Indeed, there is evi-
or Fas Antigen
dence that stimulation of some cell lines by trophic cy-
tokines or increase in their level of expression of the The APO-1 antigen was defined during studies of
bcl-2 proto-oncogene (the bcl-2 gene product inhibits monoclonal antibodies raised against a human B-lym-
apoptosis occurring in a variety of circumstances; see phoblast cell One of the antibodies was found to
section on genetic regulation) can greatly increase their induce apoptosis of activated human B- and T-lympho-
resistance to the apoptosis-inducing effect of anti- cytes and of the cells of a variety of human lymphoid
cancer d r u g ~ . ~ ~ , ' ~ ~ - ' ~ ~ 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 anti-
body 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 temper- molecule 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 simi- Injection of anti-APO-1 monoclonal antibodies
lar to that described previously for radiation and anti- causes rapid regression of murine xenografts of APO-
cancer drugs-rapidly proliferating normal cell popula- l-expressing human lymphoid cell lines, with the re-
tions, lymphoid organs, and tumors.'0~"~'6~'8~'59~'61
As is gression being accompanied by greatly enhanced apop-
the 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 an- the effect of anti-APO-1 antibodies on normal cells
other.17No definitive information is available on how would preclude their administration to humans. How-
hyperthermia 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 real- other similar receptors may yield results with therapeu-
ized until its mechanism of action is better understood. tic applications."j8

Induction of Apoptosis by Hormone Withdrawal or

Addition Induction of Apoptosis by Cytotoxic Lymphocytes

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-
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
K-cells171and NK cells'72
induced by T-cells,43,75,76,169,170
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.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 Apopto-
sis induced by cytotoxic T-cells is not blocked by inhibi-
tors of protein synthesis43or by bcl-2 e x p r e ~ s i o nDis-
.~~
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.
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 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 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 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 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
c-myc as a consequence of gene t r a n ~ f e r . ~ ~ Al- , ~ ~ , ~tute
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
, in contrast, normal cells downregulate c-
media30,32,33.
myc under these circumstances and pass into a quies-
cent state.32In 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.52In 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 o n c o g e n e ~ i s . ~ , ~ ~ , ~ ~
The possible involvement of c-fos expression in the
initiation of apoptosis has been critically reviewed.76
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.34In 1988 it was shown that
introduction of the gene into interleukin-3-dependent
2021
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 r a d i a 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-
, ~ some
~ of the most frequently encountered genetic
defects in human cancer.177This 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.77These include
growth arrest, which occurs primarily in the G I 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
apoptosi~.~*~~
To what extent p53 is involved in regulating apop-
tosis under normal conditions is ~ n k n 0 w n .Itl ~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
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.
References
1. Raff MC. Social controls on cell survival and cell death. Nature
1992; 356:397-400.
2. Williams GT. Programmed cell death: apoptosis and oncogene-
sis. Cell 1991; 65:1097-8.
3. Marx J. Cell death studies yield cancer clues. Science 1993;
259:760-1.
4 . Kerr JFR, Searle J. A suggested explanation for the paradoxically
slow growth rate of basal-cell carcinomas that contain numer-
ous mitotic figures. ] Pathol 1972; 107:41-4.
5. Kerr JFR, Wyllie AH, Cume AR. Apoptosis: a basic biologcal
phenomenon with wide-ranging implications in tissue kinetics.
Br Cancer 1972; 26:239-57.
6. Ken JFR, Searle J, Apoptosis: its nature and kinetic role. In:
Meyn RE, Withers HR, editors. Radiation biology in cancer re-
search. New York: Raven Press, 1980:367-84.
7. Stephens LC, Ang KK, Schultheiss TE, Milas L, Meyn RE. Apop-
tosis in irradiated murine tumors. Radiat Res 1991; 127:308-16.
8. Macklis RM, Lin JY, Beresford B, Atcher RW, Hines JJ, Humm JL.
Cellular kinetics, dosimetry, and radiobiology of a-particle
radioimmunotherapy: induction of apoptosis. Radiat Res 1992;
130:220-6.
9. Searle J, Lawson TA, Abbott PJ, Harmon B, Kerr JFR. An elec-
tron-microscope study of the mode of cell death induced by
cancer-chemotherapeutic agents in populations of proliferating
normal and neoplastic cells. ] Pafhol 1975; 116:129-38.
10. Dyson JED, Simmons DM, Daniel J, McLaughlin JM, Quirke P,
Bird CC. Kinetic and physical studies of cell death induced by
chemotherapeutic agents or hyperthermia. Cell Tissue Kinet
1986; 19:311-24.
11. Bany MA, Behnke CA, Eastman A. Activation of programmed
cell death (apoptosis) by cisplatin, other anticancer drugs, toxins
and hyperthermia. Biochem Pharmacol 1990; 40:2353-62.
12. Dive C, Hickman ]A. Drug-target interactions: only the first step
in the commitment to a programmed cell death? Br Cancer
1991; 641192-6.
13. Gunji H, Kharbanda 5, Kufe D. Induction of internucleosomal
DNA fragmentation in human myeloid leukemia cells by 1 -0-D-
arabinofuranosylcytosine. Cancer Res 1991; 51:741-3.
14. Cotter TG, Glynn JM, Echeverri F, Green DR. The induction of
apoptosis by chemotherapeutic agents occurs in all phases of the
cell cycle. Anticancer Res 1992; 12:773-80.
15. Johnston JB, Lee K, Verburg L, Blondal J, Mowat MRA, Israels
LG, et al. Induction of apoptosis in CD4+prolymphocytic leuke-
mia by deoxyadenosine and 2-deoxycoformycin. Leuk Res 1992;
16:781-8.
16. Harmon BV, Corder AM, Collins RJ, Gob6 GC, Allen J, Allan DJ,
et al. Cell death induced in a murine mastocytoma by 42-47C
heating in vitro: evidence that the form of death changes from
apoptosis to necrosis above a critical heat load. Znt ] Radiat Bid
1990; 58:845-58.
17. Harmon BV, Takano YS, Winterford CM, Gob6 GC. The role of
apoptosis in the response of cells and tumours to mild hyperther-
mia. Int ] Radiat Biol 1991; 59:489-501.
18. Takano YS, Harmon BV, Kerr JFR. Apoptosis induced by mild
hyperthermia in human and murine tumour cell lines: a study
using electron microscopy and DNA gel electrophoresis. Pathol
1991; 163~329-36.
19. Szende 8, Srkalovic G, Groot K, Lapis K, Schally AV. Growth
inhibition of mouse MXT mammary tumor by the luteinizing
hormone-releasing hormone antagonist SB-75. ] Natl Cancer
Inst 1990; 82:513-7.
20. Kyprianou N, English HF, Davidson NE, Isaacs JT. Programmed
cell death during regression of the MCF-7 human breast cancer
following estrogen ablation. Cancer Res 1991; 51:162-6.
21. Redding TW, Schally AV, Radulovic 5, Milovanovic 5, Szepe-
shazi K, Isaacs JT. Sustained release formulations of luteinizing
hormone-releasing hormone antagonist 58-75 inhibit prolifera-
tion and enhance apoptotic cell death of human prostate carci-
noma (PC-82) in male nude mice. Cancer Res 1992; 52:2538-44.
22. Wyllie AH, Rose KA, Morris RG, Steel CM, Foster E, Spandidos
DA. Rodent fibroblast tumours expressing human myc and ras
genes: growth, metastasis and endogenous oncogene expres-
sion. Br I Cancer 1987; 56:251-9.
23. Buttyan R, Zakeri Z, Lockshin R, Wolgemuth D. Cascade induc-
tion of c-fos, c-myc, and heat shock 70K transcripts during re-
gression of the rat ventral prostate gland. Mol Endocrinol 1988;
21650-7.
24. Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic
cell survival and cooperates with c-myc to immortalize pre-B
cells. Nature 1988; 335:440-2.
25. Hockenbery D, Nufiez G, Milliman C, Schreiber RD, Korsmeyer
SJ. Bcl-2 is an inner mitochondria1 membrane protein that
blocks programmed cell death. Nature 1990; 348:334-6.
26. Askew DS, Ashmun RA, Simmons BC, Cleveland JL. Constitu-
tive c-myc expression in an IL-3-dependent myeloid cell line
suppresses cell cycle arrest and accelerates apoptosis. Oncogene
1991; 6:1915-22.
27. Hockenbery DM, Zutter M, Hickey W, Nahm M, Korsmeyer SJ.
BCL2 protein is topographically restricted in tissues character-
ized by apoptotic cell death. Proc Natl Acad Sci USA 1991;
88:6961-5.
28. Yonish-Rouach E, Resnitzky D, Lotem J, Sachs L, Kimchi A,
Oren M. Wild-type p53 induces apoptosis of myeloid leukaemic
cells that is inhibited by interleukin-6. Nature 1991; 352:345-7.
29. Alnemri ES, Fernandes TF, Haldar 5, Croce CM, Litwack G.
Involvement of bcl-2 in glucocorticoid-induced apoptosis of hu-
man pre-8-leukemias. Cancer Res 1992; 52:491-5.
30. Bissonnette RP, Echeverri F, Mahboubi A, Green DR. Apoptotic
cell death induced by c-myc is inhibited by bcl-2. Nature 1992;
359:552-4.
31. Colotta F, Polentarutti N, Sironi M, Mantovani A. Expression
and involvement of c-fos and c-jun protooncogenes in pro-
grammed cell death induced by growth factor deprivation in
lymphoid cell lines. ] Biol Chem 1992; 267:18278-83.
32. Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks
Apoptosis in Cancer/Kerr e t al.
M, et al. Induction of apoptosis in fibroblasts by c-niyc protein.
Cell 1992; 69:119-28.
33. Fanidi A, Harrington EA, Evan GI. Cooperative interaction be-
tween c-myc and bcl-2 proto-oncogenes. Natzrre 1992; 359:
554-6.
34. Korsmeyer SJ.Bcl-2 initiates a new category of oncogenes: regu-
lators of cell death. Blood 1992; 80:879-86.
35. Miyashita T, Reed JC. Bcl-2 gene transfer increases relative resis-
tance of 549.1 and WEH17.2 lymphoid cells to cell death and
DNA fragmentation induced by glucocorticoids and multiple
chemotherapeutic drugs. Cancer Res 1992; 52:5407-11.
36. Shaw P, Bovey R, Tardy S, Sahli R, Sordat B, Costa J. Induction
of apoptosis by wild-type p53 in a human colon tumor-derived
cell line. Proc Nut2 Acad Sci USA 1992; 89:4495-9.
37. Shi Y, Glynn JM, Guilbert L], Cotter TG, Bissonnette RP, Green
DR. Role for c-myc in activation-induced apoptotic cell death in
T cell hybridomas. Science 1992; 257:212-4.
38. Vaux DL, Aguila HL, Weissman IL. Bcl-2 prevents death of fac-
tor-deprived cells but fails to prevent apoptosis in targets of cell
mediated killing. Znt Zmmunol 1992; 4:821-4.
39. Jacobson MD, Bume JF, King MI, Miyashita T, Reed JC, Raff
MC. Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA.
Nature 1993; 361:365-9.
40. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is as-
sociated with endogenous endonuclease activation. Nature
1980; 284~555-6.
41. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of
apoptosis. Int Rev Cytol 1980; 68251-306.
42. Searle J, Kerr JFR, Bishop CJ. Necrosis and apoptosis: distinct
modes of cell death with fundamentally different significance.
Pathol Annu 1982; 17(2):229-59.
43. Duke RC, Chervenak R, Cohen JJ. Endogenous endonuclease-
induced DNA fragmentation: an early event in cell-mediated
cytolysis. Proc Natl Acad Sci USA 1983; 80:6361-5.
44. Afanasev VN, Korol BA, Mantsygin YA, Nelipovich PA, Pe-
chatnikov VA, Umansky SR. Flow cytometry and biochemical
analysis of DNA degradation characteristic of two types of cell
death. FEBS Lett 1986; 194:347-50.
45. Duvall E, Wyllie AH. Death and the cell. lmmunol Today 1986;
7~115-9.
46. Wyllie AH. Cell death. Int Rev Cytol 1987; 17(Suppl):755-85.
47. Walker NI, Harmon BV, Gob6 GC, Kerr JFR. Patterns of cell
death. Methods Achien Exp Pathol 1988; 13:18-54.
48. Cohen JJ. Programmed cell death in the immune system. A d v
Znimunol 1991; 50:55-85.
49. Fesus L, Davies PJA, Piacentini M. Apoptosis: molecular mecha-
nisms in programmed cell death. Eur \ Cell Biol 1991; 56:170-7.
50. Golstein P, Ojcius DM, Young JD-E. Cell death mechanisms and
the immune system. Zmmu~iolRev 1991; 121:29-65.
51. Kerr JFR, Harmon BV. Definition and incidence of apoptosis: an
historical perspective. In: Tomei LD, Cope FO, editors. Apopto-
sis: the molecular basis of cell death. New York: Cold Spring
Harbor Laboratory Press, 1991:5-29.
52. Wyllie AH. Apoptosis and the regulation of cell numbers in nor-
mal and neoplastic tissues: an overview. Cancer Metastasis Rev
1992; 11:95-103.
53. Kerr JFR, Searle J, Harmon BV, Bishop CJ. Apoptosis. In: Potten
CS, editor. Perspectives on mammalian cell death. Oxford: Ox-
ford University Press, 19 87:9 3- 128.
54. Arends MJ, Wyllie AH. Apoptosis: mechanisms and roles in pa-
thology. lnt Rev Exp Pathol 1991; 32223-54.
55. Bursch W, PaffeS, Putz B, Barthel G, Schulte-Hermann R. Deter-
mination of the length of the histological stages of apoptosis in
2023
normal liver and in altered hepatic foci of rats. Carcinogenesis
1990; 11:847-53.
56. Kyprianou N, Isaacs JT. Activation of programmed cell death in
the rat ventral prostate after castration. Eiidocrinology 1988;
122:552-62.
57. Rotello RJ, Hocker MB, Gerschenson LE. Biochemical evidence
for programmed cell death in rabbit uterine epithelium. A m 1
Path02 1989; 134:491-5.
58. McConkey DJ, Aguilar-Santelises M, Hartzell P, Eriksson 1,
Mellstedt H, Orrenius S, et al. Induction of DNA fragmentation
in chronic B-lymphocytic leukemia cells. \ Zmmunol 1991;
146: 1072-6.
59. Yanagihara K, Tsumuraya M. Transforming growth factor &
induces apoptotic cell death in cultured human gastric carci-
noma cells. Cancer Res 1992; 52:4042-5.
60. Cohen GM, Sun X-M, Snowden RT, Dinsdale D, Skilleter DN.
Key morphological features of apoptosis may occur in the ab-
sence of internucleosomal DNA fragmentation. Biochem 1992;
2861331-4.
61. Collins RJ, Harmon BV, Gob6 GC, Kerr JFR. Intemucleosomal
DNA cleavage should not be the sole criterion for identifying
apoptosis. Znt \ Radiat B i d 1992; 61:451-3.
62. Ucker DS, Obermiller PS, Eckhart W, Apgar JR, Berger NA,
Meyers J. Genome digestion is a dispensable consequence of
physiological cell death mediated by cytotoxic T lymphocytes.
Mol Cell Biol 1992; 12:3060-9.
63. Oberhammer F, Wilson JW, Dive C, Moms ID, Hickman JA,
Wakeling AE, et al. Apoptotic death in epithelial cells: cleavage
of DNA to 300 and/or 50 kb fragments prior to or in the absence
of internucleosomal fragmentation. EMBO \ 1993; 123679-84.
64. Cohen JJ, Duke RC. Glucocorticoid activation of a calcium-de-
pendent endonuclease in thymocyte nuclei leads to cell death. ]
Zmmunol 1984; 13238-42.
65. Alnemri ES, Litwack G. Activation of intemucleosomal DNA
cleavage in human CEM lymphocytes by glucocorticoid and no-
vobiocin: evidence for a non-Ca2+-requiring mechanism(s). \
Biol Chem 1990; 265:17323-33.
66. Arends MJ, Moms RG, Wyllie AH. Apoptosis: the role of the
endonuclease. Am \ Pathol 1990; 136:593-608.
67. Caron-Leslie L-AM, Schwartzman RA, Gaido ML, Compton
MM, Cidlowski JA. Identification and characterization of gluco-
corticoid-regulated nuclease(s) in lymphoid cells undergoing
apoptosis. J Steroid Biocliem Mol Biol 1991; 40:661-71.
68. Gaido ML, Cidlowski JA. Identification, purification, and charac-
terization of a calcium-dependent endonuclease (NUCIS) from
apoptotic rat thymocytes: NUC18 is not histone H,B. Biol Chem
1991; 266: 18580-5.
69. Barry MA, Eastman A. Identification of deoxyribonuclease I1 as
an endonuclease involved in apoptosis. Arch Biochem Biophys
1993; 300:440-50.
70. Peitsch MC, Polzar B, Stephan H, Crompton T, MacDonald HR,
Mannherz HG, et al. Characterization of the endogenous deoxy-
ribonuclease involved in nuclear DNA degradation during
apoptosis (programmed cell death). EMBO ] 1993; 12371-7.
71. Murgia M, Pizzo P, Sandoni D, Zanovello P, Rizzuto R, Di Vir-
gilio F. Mitochondria1 DNA is not fragmented during apoptosis.
\ B i d Chem 1992; 267:10939-41.
72. Tepper CG, Studzinski GP. Teniposide induces nuclear but not
mitochondrial DNA degradation. Cancer Res 1992; 52:3384-90.
73. Wyllie AH, Morris RG. Hormone-induced cell death: purifica-
tion and properties of thymocytes undergoing apoptosis after
glucocorticoid treatment. A m ] Pathol 1982; 109:78-87.
2024 CANCER April 15, 1994, Volume 7 3 , No. 8
74.Russell SW, Rosenau W, Lee JC. Cytolysis induced by human
lymphotoxin: cinemicrographic and electron microscopic obser-
vations. An7 ] Pathol 1972; 69:103-18.
75. Sanderson CJ. The mechanism of Tcell mediated cytotoxicity: 11.
morphological studies of cell death by time-lapse microcinema-
tography. Proc R Soc Lond (Bid) 1976; 192241-55.
76. Matter A. Microcinematographic and electron microscopic analy-
sis of target cell lysis induced by cytotoxic T lymphocytes. Immu-
l~ology1979; 36:179-90.
77.Pittman SM, Geyp M, Tynan SJ, Gramacho CM, Strickland DH,
Fraser MJ, et al. Tubulin in apoptotic cells. In: Lavin M, Watters
D, editors. Programmed cell death: the cellular and molecular
biology of apoptosis. Switzerland: Harwood Academic Pub-
lishers, 1993:315-23.
78. Cotter TG, Lennon SV, Glynn JM, Green DR. Microfilament-
disrupting agents prevent the formation of apoptotic bodies in
tumor cells undergoing apoptosis. Cancer Res 1992; 52:997-
1005.
79.Fesus L, Thomazy V, Falus A. Induction and activation of tissue
transglutaminase during programmed cell death. FEBS Lett
1987;224:104-8.
80. Fesus L, Thomazy V. Searching for the function of tissue trans-
glutaminase: its possible involvement in the biochemical path-
way of programmed cell death. Adv Exp Med Biol 1988;
231 :I 19-34.
81.Fesus L, Thomazy V, Autuori F, Ceru MI, Tarcsa E, Piacentini
M. Apoptotic hepatocytes become insoluble in detergents and
chaotropic agents as a result of transglutaminase action. FEBS
Lett 1989; 245:150-4.
82.Piacentini M, Fesus L, Farrace MG, Ghibelli L, Piredda L, Melino
G. The expression of tissue transglutaminase in two human
cancer cell lines is related with programmed cell death (apopto-
sis). Eur ] Cell Biol 1991;54:246-54.
83.Piacentini M, Autuori F, Dini L, Farrace MG, Ghibelli L, Piredda
L, et al. Tissue transglutaminase is specifically expressed in
neonatal rat liver cells undergoing apoptosis upon epidermal
growth factor-stimulation. Cell Tissue Res 1991;263:227-35.
84.Tenniswood MP, Guenette RS, Lakins J, Mooibroek M, Wong P,
Welsh J-E. Active cell death in hormone-dependent tissues.
Caticer Metastasis Rev 1992; 11:197-220.
85.Savill J,Fadok V, Henson P, Haslett C. Phagocyte recognition of
cells undergoing apoptosis. Immunol Today 1993;14:131-6.
86.Duvall E, Wyllie AH, Morns RG. Macrophage recognition of
cells undergoing programmed cell death (apoptosis). Zmmunol-
O ~ 1985;
Y 56:351-8.
87. Savill J, Dransfield I, Hogg N, Haslett C. Vitronectin receptor-
mediated phagocytosis of cells undergoing apoptosis. Nature
1990; 343:170-3.
88. Savill J, Hogg N, Ren Y, Haslett C. Thrombospondin cooperates
with CD36 and the vitronectin receptor in macrophage recogni-
tion of neutrophils undergoing apoptosis. J Clin Invest 1992;
90:1513-22.
89. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL,
Henson PM. Exposure of phosphatidylserine on the surface of
apoptotic lymphocytes triggers specific recognition and removal
by macrophages. J lmmunol 1992; 148:2207-16.
90. Buttyan R, Olsson CA, Pintar J, Chang C, Bandyk M, Ng P-Y, et
al. Induction of the TRPM-2 gene in cells undergoing pro-
grammed death. Mol Cell Biol 1989;9:3473-81.
91.Pearse MJ, OBryan M, Fisicaro N, Rogers L, Murphy B, dApice
AJ. Differential expression of clusterin in inducible models of
apoptosis. int Immunol 1992; 4:1225-31.
92. Owens GP, Hahn WE, Cohen JJ. Identification of mRNAs asso-
ciated with programmed cell death in immature thymocytes.
Mol Cell Biol 1991;11:4177-88.
93. Owens GP, Cohen JJ. Identification of genes involved in pro-
grammed cell death. Cancer Metastasis Rev 1992; 11:149-56.
94. Ishida Y, Agata Y, Shibahara K, Honjo T. Induced expression of
PD-1, a novel member of the immunoglobulin gene superfa-
mily, upon programmed cell death. EMBO J 1992; 11:3887-95.
95. Wyllie AH, Morris RG, Smith AL, Dunlop D. Chromatin cleav-
age in apoptosis: association with condensed chromatin mor-
phology and dependence on macromolecular synthesis. J Pathol
1984;142~67-77.
96. Sellins KS, Cohen JJ. Gene induction by y-irradiation leads to
DNA fragmentation in lymphocytes. J Znimunol 1987;
139~3 199-206.
97. Shi Y, Szalay MG, Paskar L, Boyer M, Singh B, Green DR. Acti-
vation-induced cell death in T cell hybridomas is due to apopto-
sis. J Immutiol 1990; 144:3326-33.
98. Crompton T. IL3-dependent cells die by apoptosis on removal
of their growth factor. Growth Factors 1991;4:109-16.
99. Waring P. DNA fragmentation induced in macrophages by glio-
toxin does not require protein synthesis and is preceded by
raised inositol triphosphate levels. ] Biol Chem 1990;265:14476-
80.
100. Martin SJ, Lennon SV, Bonham AM, Cotter TG. Induction of
apoptosis (programmed cell death) in human leukemic HL-60
cells by inhibition of RNA or protein synthesis. J lmmunol 1990;
145:1859-67.
101. Collins RJ,Harmon BV, Souvlis T, Pope JH, Kerr JFR. Effects of
cycloheximide on B-chronic lymphocytic leukaemic and normal
lymphocytes in vitro: induction of apoptosis. Br J Cancer 1991;
64:518-22.
102. Bansal N, Houle A, Melnykovych G: Apoptosis: mode of cell
death induced in T cell leukemia lines by dexamethasone and
other agents. FASEBJ 1991;5:211-6.
103. McConkey DJ, Hartzell P, Duddy SK, Hikansson H, Orrenius S.
2,3,7,8-tetrachlorodibenzo-p-dioxinkills immature thymocytes
by Ca2+-mediated endonuclease activation. Science 1988;
242:256-9.
104.Ucker DS, Ashwell JD, Nickas G. Activation-driven T cell death:
1. requirements for de novo transcription and translation and
association with genome fragmentation. J Immunol 1989;
143~3461-9.
105. Ferrer I. The effect of cycloheximide on natural and x-ray-in-
duced cell death in the developing cerebral cortex. Brain Res
1992; 588:351-7.
106. McConkey DJ, Orrenius S, Jondal M. Cellular signalling in pro-
grammed cell death (apoptosis). Immunol Today 1990;11:120-1.
107. Bansal N, Houle AG, Melnykovych G. Dexamethasone-induced
killing of neoplastic cells of lymphoid derivation: lack of early
calcium involvement. J Cell Physiol 1990; 143:105-9.
108.Lennon SV, Kilfeather SA, Hallett MB, Campbell AK, Cotter
TG. Elevations in cytosolic free Ca2+are not required to trigger
apoptosis in human leukaemia cells. Clin Exp Immunol 1992;
87:465-71.
109.Dive C, Evans CA, Whetton AD. Induction of apoptosis: new
targets for cancer chemotherapy. Semiti Cancer Biol 1992;3:417-
27.
110. Kerr JFR, Harmon B, Searle J. An electron-microscope study of
cell deletion in the anuran tadpole tail duringspontaneous meta-
morphosis with special reference to apoptosis of striated muscle
fibres. J Cell Sci 1974;14:571-85.
1 1 1 . Kerr JFR. Shrinkage necrosis: a distinct mode of cellular death. J
Pathol 1971; 105:13-20.
Apoptosis in Cancer/Kerr et al.
112. Benedetti A, JGzGquelAM, Orlandi F. Preferential distribution of
apoptotic bodies in acinar zone 3 of the normal human and rat
liver. ] Hepatol 1988; 7:319-24.
113. Kerr JFR, Searle J. Deletion of cells by apoptosis during castra-
tion-induced involution of the rat prostate. Virchows Arch (Cell
Patholl 1973; 13:87-102.
114. Wyllie AH, Kerr JFR, Macaskill IAM, Currie AR. Adrenocortical
cell deletion: the role of ACTH. ] Pafhol 1973; 111:85-94.
115. Potten CS. Extreme sensitivity of some intestinal crypt cells to X
and y irradiation. Nature 1977; 269:518-21.
116. Allan DJ, Harmon BV, Kerr JFR. Cell death in spermatogenesis.
In: Potten CS, editor. Perspectives on mammalian cell death.
Oxford: Oxford University Press, 1987:229-58.
117. Sandow BA, West NB, Norman RL, Brenner RM. Hormonal
control of apoptosis in hamster uterine luminal epithelium. A m 1
Atiat 1979; 156:15-36.
118. Duke RC, Cohen JJ. IL-2 addiction: withdrawal of growth factor
activates a suicide program in dependent T cells. Lymphokine Res
1986; 5:289-99.
119. Williams GT, Smith CA, Spooncer E, Dexter TM, Taylor DR.
Haemopoietic colony stimulating factors promote cell survival
by suppressing apoptosis. Nature 1990; 343:76-9.
120. Araki S, Shimada Y, Kaji K, Hayashi H. Apoptosis of vascular
endothelial cells by fibroblast growth factor deprivation. Bio-
chem Biophys Res Commuri 1990; 168:1194-200.
121. Mangan DF, Wahl SM. Differential regulation of human mono-
cyte programmed cell death (apoptosis) by chemotactic factors
and proinflammatory cytokines. ] Immunol 1991; 147:3408-12.
122. Rodriguez-Tarduchy G, Collins MKL, Garcia I, L6pez-Rivas A.
Insulin-like growth factor I inhibits apoptosis in IL-3-depen-
dent hemopoietic cells. ] Immunol 1992; 149:535-40.
123. Lynch MP, Nawaz S, Gerschenson LE. Evidence for soluble fac-
tors regulating cell death and cell proliferation in primary cul-
tures of rabbit endometrial cells grown on collagen. Proc Natl
Acad Sci USA 1986; 83:4784-8.
124. Walker NI, Bennett RE, Kerr JFR. Cell death by apoptosis during
involution of the lactating breast in mice and rats. A m ] Anat
1989; 185:19-32.
125. OShea JD, Hay MF, Cran DG. Ultrastructural changes in the
theca interna during follicular atresia in sheep. ] Reprod Fertil
1978; 54:183-7.
126. Hughes FM, Gorospe WC. Biochemical identification of apopto-
sis (programmed cell death) in granulosa cells: evidence for a
potential mechanism underlying follicular atresia. Eiidocriiiology
1991; 129:2415-22.
127. Weedon D, Strutton G. Apoptosis as the mechanism of the invo-
lution of hair follicles in catagen transformation. Acfa Derm Ven-
ereol (Stockh) 1981; 61:335-9.
128. Smith CA, Williams GT, Kingston R, Jenkinson EJ, Owen JJT.
Antibodies to CD3/T-cell receptor complex induce cell death by
apoptosis in immature T cells in thymic cultures. Nature 1989;
337~181-4.
129. Liu Y-J, Joshua DE, Williams GT, Smith CA, Gordon J, MacLen-
nan ICM. Mechanism of antigen-driven selection in germinal
centres. Nature 1989; 342:929-31.
130. Savill IS, Wyllie AH, Henson JE, Walport MJ, Henson PM, Has-
lett C. Macrophage phagocytosis of aging neutrophils in inflam-
mation: programmed cell death in the neutrophil leads to its
recognition by macrophages. ] Clin Invest 1989; 83:865-75.
131. Radley JM, Haller CJ. Fate of senescent megakaryocytes in the
bone marrow. B r Haematol 1983; 53:277-87.
132. Searle J, Collins DJ, Harmon B, Kerr JFR. Thespontaneous occur-
rence of apoptosis in squamous carcinomas of the uterine cervix.
Pathology 1973; 5:163-9.
2025
133. Wyllie AH. The biology of cell death in tumours. Aitticaiicer Res
1985; 5~131-6.
134. Harmon BV. An ultrastructural study of spontaneous cell death
in a mouse mastocytoma with particular reference to dark cells. ]
Pathol 1987; 153:345-55.
135. Sarraf CE, Bowen ID. Proportions of mitotic and apoptotic cells
in a range of untreated experimental tumours. Cell Tissue Kiiiet
1988; 21:45-9.
136. Gob6 GC, Axelsen RA, Searle JW. Cellular events in experimen-
tal unilateral ischemic renal atrophy and in regeneration after
contralateral nephrectomy. Lab Invest 1990; 63:770-9.
137. Bellomo G, Perotti M, Taddei F, Mirabelli F, Finardi G, Nicotera
P, et al. Tumor necrosis factor a induces apoptosis in mammary
adenocarcinoma cells by an increase in intracellular free Ca2+
concentration and DNA fragmentation. Cancer Res 1992;
52~1342-6.
138. Wright SC, Kumar P, Tam AW, Shen N, Varma M, Larrick JW.
Apoptosis and DNA fragmentation precede TNF-induced cyto-
lysis in U937 cells. ]Cell Biochem 1992; 48:344-55.
139. Curson C, Weedon D. Spontaneous regression in basal cell carci-
nomas. Cutan Pathol 1979; 6:432-7.
140. Bursch W, Lauer B, Timmermann-Trosiener I, Barthel G,
Schuppler J, Schulte-Hermann R. Controlled death (apoptosis)
of normal and putative preneoplastic cells in rat liver following
withdrawal of tumor promoters. Carciriogenesis 1984; 5:453-8.
41. Columbano A, Ledda-Columbano GM, Rao PM, Rajalakshmi 5,
Sarma DSR. Occurrence of cell death (apoptosis) in preneoplas-
tic and neoplastic liver cells: a sequential study. A m ] Pathol
1984; 116:441-6.
42. Gob6 GC, Axelsen RA, Harmon BV, Allan DJ. Cell death by
apoptosis following X-irradiation of the foetal and neonatal rat
kidney. Int ] Radiat Biol 1988; 54:567-76.
43. Trowell OA. Ultrastructural changes in lymphocytes exposed to
noxious agents in vitro. Q ] Exp Physiol 1966; 51:207-20.
44. Yamada T, Ohyama H. Radiation-induced interphase death of
rat thymocytes is internally programmed (apoptosis). Znt ] Radiat
Biol 1988; 53:65-75.
45. Cohen JJ, Duke RC. Apoptosis and programmed cell death in
immunity. Annu Rev Immunol 1992; 10:267-93.
146. Sodicoff M, Pratt NE, Sholley MM. Ultrastructural radiation in-
jury of rat parotid gland: a histopathologic dose-response study.
Radiat Res 1974; 58:196-208.
147. Stephens LC, Schultheiss TE, Small SM, Ang KK, Peters LJ. Re-
sponse of parotid gland organ culture to radiation. Radiat Res
1989; 120:140-53.
148. Gazda MJ, Schultheiss TE, Stephens LC, Ang KK, Peters LJ. The
relationship between apoptosis and atrophy in the irradiated
lacrimal gland. Int ] Radiat olicol Biol Phys 1992; 24:693-7.
149. Falkvoll KH. Quantitative histological changes in a human mela-
noma xenograft following exposure to single dose irradiation
and hyperthermia. Int ] Radiat Oncol Biol Phys 1991; 21:989-94.
150. Forster TH, Allan DJ, Gob6 GC, Harmon BV, Walsh TP, Kerr
JFR. Beta-radiation from tracer doses of 32Pinduces massive
apoptosis in a Burkitts lymphoma cell line. lnt ] Radiat Biol
1992; 61~365-7.
151. Lane DP. p53, guardian of the genome. Nature 1992; 358:15-6.
152. Clarke AR, Purdie CA, Harrison DJ, Morris RG, Bird CC,
Hooper ML, et al. Thymocyte apoptosis induced by p53-depen-
dent and independent pathways. Nature 1993; 362:849-52.
153. Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T. p53 is
required for radiation-induced apoptosis in mouse thymocytes.
Nature 1993; 3622347-9.
154. Roy C, Brown DL, Little JE, Valentine BK, Walker PR, Sikorska
M, et al. The topoisomerase I1 inhibitor teniposide (VM-26) in-
2026 CANCER April 25, 2994, Volume 73, No. 8
duces apoptosis in unstimulated mature murine lymphocytes.
Exp Cell Res 1992; 200:416-24.
155. Hickman JA. Apoptosis induced by anticancer drugs. Cancer
Metastasis Rev 1992; 11:121-39.
156. Collins MKL, Marvel J, Malde P, Lopez-Rivas A. Interleukin 3
protects murine bone marrow cells from apoptosis induced by
DNA damaging agents. ] Exp Med 1992; 176:1043-51.
157. Lotem J, Sachs L. Hematopoietic cytokines inhibit apoptosis in-
duced by transforming growth factor pl and cancer chemother-
apy compounds in myeloid leukemic cells. Blood 1992; 80:
1750-7.
158. Fisher TC, Milner AE, Gregory CD, Jackman AL, Aherne GW,
Hartley JA, et al. Bcl-2 modulation of apoptosis induced by anti-
cancer drugs: resistance to thymidylate stress is independent of
classical resistance pathways. Cancer Res 1993; 53:3321-6.
159. Wanner RA, Edwards MJ, Wright RG. The effect of hyperther-
mia on the neuro-epithelium of the 21-day guinea-pig foetus:
histologic and ultrastructural study. ] Pathol 1976; 118:235-44.
160. Allan DJ, Harmon BV. The morphologic categorization of cell
death induced by mild hyperthermia and comparison with
death induced by ionizing radiation and cytotoxic drugs. Scan
Electron Microsc 1986; 3:1121-33.
161. Sellins KS, Cohen JJ. Hyperthermia induces apoptosis in thymo-
cytes. Radiat Res 1991; 126:88-95.
162. Galili U, Leizerowitz R, Moreb J, Gamliel H, Gurfel D, Polliack
A. Metabolic and ultrastructural aspects of the in vitro lysis of
chronic lymphocytic leukemia cells by glucocorticoids. Cancer
Res 1982; 42:1433-40.
163. McDonnell TJ, Troncoso P, Brisbay SM, Logothetis C, Chung
LWK, Hsieh J-T, et al. Expression of the protooncogene bcl-2 in
the prostate and its association with emergence of androgen-in-
dependent prostate cancer. Cancer Res 1992; 52:6940-4.
164. Trauth BC, Klas C, Peters AMJ, Matzku S, Moller P, Falk W, et
al. Monoclonal antibody-mediated tumor regression by induc-
tion of apoptosis. Science 1989; 245:301-5.
165. ltoh N, Yonehara S, lshii A, Yonehara M, Mizushima S-I, Same-
shima M, et al. The polypeptide encoded by the cDNA for hu-
man cell surface antigen Fas can mediate apoptosis. Cell 1991;
66~233-43.
166. Oehm A, Behrmann I, Falk W, Pawlita M, Maier G, Klas C, et al.
Purification and molecular cloning of the APO-1 cell surface
antigen, a member of the tumor necrosis factor/nerve growth
factor receptor superfamily: sequence identity with the Fas anti-
gen. ] Biol Chem 1992; 267:10709-15.
167. Krammer PH, Behrmann I, Bier V, Daniel P, Dhein J, Falk MH,
et al. Apoptosis in the APO-I system. In: Tomei LD, Cope FO,
editors. Apoptosis: the molecular basis of cell death. New York:
Cold Spring Harbor Laboratory Press, 1991237-99.
168. Dhein J, Daniel PT, Trauth BC, Oehm A, Moller P, Krammer
PH. Induction of apoptosis by monoclonal antibody anti-APO-1
class switch variants is dependent on cross-linking of APO-1
cell surface antigens. ] Immunol 1992; 149:3166-73.
169. Don MM, Ablett G, Bishop CJ, Bundesen PG, Donald KJ, Searle
J, et al. Death of cells by apoptosis following attachment of spe-
cifically allergized lymphocytes in vitro. Aust Exp Biol Med Sci
1977; 55:407-17.
170. Russell JH, Masakowski V, Rucinsky T, Phillips G. Mechanisms
of immune lysis I11 characterization of the nature and kinetics of
the cytotoxic T lymphocyte-induced nuclear lesion in the target.
] Immuiiol 1982; 128:2087-94.
171. Sanderson CJ, Thomas JA. The mechanism of K cell (antibody-
dependent) cell mediated cytotoxicity 11: characteristics of the
effector cell and morphological changes in the target cell. Proc R
SOC Lor~d(Biol) 1977; 197~417-24.
172. Bishop CJ, Whiting VA. The role of natural killer cells in the
intravascular death of intravenously injected murine tumour
cells. Br ] Cancer 1983; 48:441-4.
173. Searle J, Kerr JFR, Battersby C, Egerton WS, Balderson G, Bur-
nett W. An electron microscopic study of the mode of donor cell
death in unmodified rejection of pig liver allografts. Aust ] Exp
Biol Med Sci 1977; 55:401-6.
174. Martz E, Howell DM. CTL: virus control cells first and cytolytic
cells second? DNA fragmentation, apoptosis and the prelytic
halt hypothesis. Immunol Today 1989; 10:79-86.
175. Shi L, Kam C-M, Powers JC, Aebersold R, Greenberg AH. Purifi-
cation of three cytotoxic lymphocyte granule serine proteases
that induce apoptosis through distinct substrate and target cell
interactions. ] Exp M e d 1992; 176:1521-9.
176. Smeyne RJ, Vendrell M, Hayward M, Baker SJ, Mia0 GG, Schil-
ling K, et al. Continuous c-fos expression precedes programmed
cell death in vitro. Nature 1993; 363:166-9.
177. Oren M. p53: the ultimate tumor suppressor gene? FASEB] 1992;
6:3169-76.
178. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgo-
mery CA, Butel JS, et al. Mice deficient for p53 are developmen-
tally normal but susceptible to spontaneous tumours. Nature
1992; 356:2 15-21.
179. Lane DP. A death in the life of p53. Nature 1993; 362786-7.