Apoptosis John F. R. Kerr, Ph.D.,* Clay M. Winterford

2013

Apoptosis Its Significance in Cancer and Cancer Therapy

John F. R. Kerr, Ph.D.,* Clay M. Winterford, Assoc.Dipl.Appl.Biol.,* and Brian V. Harmon, Ph.D.t

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

From the 'Department of Pathology, University of Queensland Medical School, Herston, Queensland; and the tSchool of Life Science, Queensland University of Technology, Brisbane, 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 deletion, DNA fragmentation, radiation, anti-cancer drugs, hyperthermia, hormone ablation, proto-oncogene, tumor suppressor gene.

Oncologists traditionally have been concerned primarily with cell proliferation. However, apoptosis (the dis- tinctive form of cell death that complements cell proliferation in normal tissue homeostasis)' increasingly has been attracting their attention.'f3

The realization that apoptosis occurs in tumors is not new. More than 20 years ago it was suggested that apoptosis may account for much of the spontaneous cell loss known from kinetic studies to occur in many

and it has been clear for some time that its extent often is enhanced in tumors by well-established treatment modalities, such as cytotoxic chern~therapy:-'~ heating,'0~"~'6-'8 and hormone ablation.l9-'' However, during the past few years, advances in understanding of the control of apoptosis at the molecular level have extended its potential 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 products of certain proto-oncogenes and the p53 tumor suppressor has opened up exciting avenues for future re~earch.~

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

Figure 2. Apoptosis of murine NS-I myeloma cell occurring spontaneously in culture. Note the sharply delineated masses of condensed chromatin in membrane-enclosed nuclear fragments and remnant of nucleolus (arrow). Ribosomes are well preserved (electron micrograph, original magnification X 17,500).

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

statement with lists of references but will give a compos- ite overview; supporting bibliographies and many additional illustrations can be found in comprehensive re- views.41.53,54

The contrasting ultrastructural features of apoptosis and necrosis are shown in stylized form in Figure 1.

Apoptosis characteristically affects scattered single cells, not groups of adjoining cells, as is the case with necrosis. The earliest recognized morphologic changes are compaction and segregation of the nuclear chromatin, with the formation of sharply delineated, uniformly finely granular masses that become marginated against the nuclear envelope, and condensation of the cytoplasm. Progression of the condensation is accompanied by convo~u~on of the nuclear and cell outlines, and this is into discrete fragments that are surrounded by a double-layered envelope (Fig. 2) and by budding of the cell as a whole

Figure 3. Same culture as that illustrated in Figure 2. Separation of surface protuberances is leading to apoptotic body formation. Some nuclear fragments show peripheral masses of condensed chromatin, whereas others are uniformly dense in plane of section (electron micrograph, original magnification X13,900).

by breaking up Of the

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 preserved (Figs. 4 and 5).

Apoptotic bodies arising in tissues are quickly in- gested by nearby cells and degraded within their lysosomes (Fig. 6) . There is no associated inflammation with the outpouring of specialized phagocytes into the tissue, such as occurs with necrosis, and various types of resident cells, including epithelial cells (Fig. 6) , 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 macrophages. However, apoptotic bodies formed in cell cultures mostly escape phagocytosis and eventually degenerate.

The early cellular events in apoptosis are accom- plished quickly, with only a few minutes elapsing between 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 relatively inconspicuous by light microscopic study (Fig. 7). After phagocytosis, their digestion is completed within h o ~ r s . ~ ~ , ~ ~ This fact should be borne in mind when apoptosis is being quantified histologically.

The distinction between apoptosis and necrosis is unequivocal at the level of electron microscopic study (Fig. l), and with practice, the two processes can be recognized with confidence using light microscopic study alone. Condensation of nuclear chromatin occurs in the early stages of necrosis, but the chromatin is not radically redistributed, as it is in apoptosis, and the edges of the chromatin clumps tend to be irregular and poorly defined (Fig. 8). In addition, the nucleus of the necrotic cell never separates into discrete, 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 additional confirmatory evidence of categorization of the cell death present in a particular circumstance as necrosis, In tumors, such foci of confluent necrosis typically tend to be located in the centers of nodules, whereas individual cells undergoing apoptosis are observed throughout the viable tumor tissue (Fig. 7).

Figure 8. Spontaneous necrosis occurring in center of murine P-815 mastocytoma growing in muscle. Note the irregular clumping of chromatin and disintegration of organelles and plasma membrane (electron micrograph, original magnification X23,lOO).

Figure 9. Agarose gel electrophoresis of DNA extracted from cultures of P-815 cells. Ethidium bromide stain photographed in ultraviolet light. Lane 1: DRIgest 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 distinct entity,5 some years elapsed before significant pro- gress was made in elucidating its biochemical mechanism. In 1980, Wyllie4' showed that glucocorticoid-induced death of thymocytes, which was known to display the typical ultrastructural features of apoptosis, is associated with a unique change in the nuclear DNA. There is double-strand cleavage at the linker regions between nucleosomes, leading to the formation of fragments that are multiples of units comprising 180-200 base pairs. These fragments are detected readily by agarose gel electrophoresis, a characteristic ladder being evident when ethidium bromide-stained gels are viewed in ultraviolet light. Figure 9 shows such a ladder produced by electrophoresis of DNA extracted from apoptotic tumor cells. However, in necrosis there usually is random cleavage of DNA and degradation of h i ~ t o n e , ~ ~ * * ~ a diffuse smear developing on DNA electrophoresis (Fig. 9). Internucleosomal cleavage has been shown to accompany apoptosis occurring in a wide variety of cell types,43,56-59 and DNA electrophoresis is used extensively for identifying the process. The cleavage occasionally may be delayed or absent in cell death that appears by other criteria to be apoptotic.60-62 It has been found that internucleosomal cleavage is preceded by cleavage of DNA into 300- or 50-kilo base-pair frag-


ments in cells undergoing apoptosis and that the formation of these large DNA fragments occurs in at least some cases in which there is no subsequent development of oligonucleosomes.63 The identity of the enzyme or enzymes responsible for the internucleosomal cleavage 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 function in preventing the transfer of potentially active genetic material to nearby cells when apoptotic bodies are phagocytosed.66

The cytoplasmic condensation, which is such a prominent ultrastructural feature of apoptosis, is accompanied by an increase in cellular density." However, nothing is known about the mechanisms involved. The formation of the cell surface protuberances observed by electron microscopic study has been shown by 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 bodies, clearly suggests major participation of cytoskeletal elements. In cells destined to undergo apoptosis, p-tubulin messenger RNA increases before the development of morphologic changes and the occurrence of DNA cleavage.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 nuclear 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 separation of discrete apoptotic bodies from the condensing cell was found to coincide with abrupt cessation of cell surface movement. A probable explanation for this 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 formation of a highly cross-linked, rigid framework within apoptotic bodies, which aids in maintaining their integrity and thus in preventing leakage of their contents into the extracellular space. In several types of cells, the increase in tissue transglutaminase activity associated with apoptosis has been shown to be preceded by an increase in the level of the corresponding messenger RNA. 4934

The rapid phagocytosis of apoptotic bodies by nearby cells while their membranes are intact implies the operation of a highly specific recognition mechanism, and there is evidence that more than one such mechanism may exist.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 exposed on the surface of the apoptotic bodies. More recently, macrophage vitronectin receptors have been implicated in the recognition of neutrophil leukocytes undergoing a p o p t o s i ~ , ~ ~ , ~ ~ and evidence has been produced that the exposure of phosphatidylserine on the surface of apoptotic thymocytes and lymphocytes may lead to their specific recognition by 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 apoptosis is enhanced." However, the association does not appear to be invariable.84z91 Recently, additional putative apoptosis-related genes have been identified using subtractive hybridization technique^.^*-^^ Elucidation of the functions of their protein products is awaited.

The occurrence of apoptosis in a number of circumstances has been shown to be suppressed by inhibitors of messenger RNA or protein synthesis, such as actino-

ever, in other situations these inhibitors have no blocking 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 addition, to compound the problem, actinomycin D and cycloheximide have been shown to induce apoptosis in some normal and neoplastic cell populations.9~'00-'02 The significance of these conflicting findings is uncertain.

Theoretically, newly synthesized proteins might be required for initiation of apoptosis by certain stimuli,

mycin D and cycloheximide, r e s p e c t i ~ e l y . ~ ~ , ~ ~ - ~ ~ HOW-


for execution of the process, or for both. In the case of some triggering stimuli, there is evidence that cycloheximide exerts its blocking effect at the level of initiation,'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 protein synthetic requirement. However, evidence has been presented in the preceding paragraphs that protein 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 synthesis, the cells must possess all of the machinery necessary for execution of the death sentence. Cohen also suggests 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 apoptosis induced by certain stimuli, but cycloheximide increases apoptosis above baseline levels in the same populations when the agent is administered a l ~ n e . ' ~ ~ , ' ~ ~

Finally, there is a possible role for elevation of cytosolic Ca2+ in triggering apoptosis. There is good evidence 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 certain 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, being 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 development 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 epithelium 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 trigger apoptosis also may be involved in normal cell population homeostasis. In primary cultures of rabbit endometrial cells, factors that induce mitosis and apoptosis, 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 associated with marked enhancement of apoptosis; well- documented examples include reversion of the lactating breast to its resting state after weaning,lZ4 ovarian follicular a t r e ~ i a , ' ~ ~ ~ ' ~ ~ and catagen involution of hair folli- c l e ~ . " ~ The trigger responsible for the increased apoptosis 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 responsible 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 cytoplasm during the formation of platelet^.'^'

Spontaneous Occurrence of Apoptosis in Tumors

Apoptosis can be found in virtually all untreated malignant 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 approaches that seen in rapidly involuting tissue^,^ indi-


cating that its kinetic significance must sometimes be considerable.

The factors responsible for the spontaneous occurrence of apoptosis in tumors undoubtedly are diverse. Apoptosis often is particularly prominent near foci of confluent necrosis, where mild ischemia is likely to be involved in its initiation; this is a known cause of enhancement of apoptosis in non-neoplastic tissues."'"36 Tumor necrosis factor a has been shown to induce apoptosis in tumor cell lines in ~ i t r o , ' ~ ~ , ' ~ ~ so some of the apoptosis observed in tumors in vivo may be attrib- utable to release of this cytokine by infiltrating macrophages. In other instances, apoptosis may be a result of attack on the tumor by cytotoxic T- lymphocyte^.'^' However, increased apoptosis also is observed in preneoplastic foci and nodules developing in the liver after administration of chemical carcinogen^^^"^^^^; it is 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 carcinogenesis, 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 processes intrinsic to the tumor cells, with differing rates of apoptosis being found in otherwise similar tumors expressing different oncogenes."

Induction of Apoptosis by Radiation

Ionizing radiation, when given in small to moderate doses, greatly enhances apoptosis in certain normal tissues without producing necrosis. Cells in the stem cell region of hierarchically arranged rapidly proliferating populations such as gut ~ r y p t s , ~ , " ~ differentiating spermatogonia,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 mutant cell in a proliferating zone in the fetus would give rise to many mutant progeny in the resulting mature tissue; surviving mutant spermatogonia would give rise to mutant gametes; and some lymphocytes with mutations in their receptor genes might have the capacity to produce autoimmune disease.'45 Of course, the occurrence 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 during evolution. Nevertheless, the differentiated acinar cells of lacrimal and salivary glands have been found to be susceptible to the induction of apoptosis by radiation, although bigger doses are req~ired. '~~- '~ ' This susceptibility is not readily explicable on the basis of the teleologic argument put forward.

There have been surprisingly few studies of apoptosis in irradiated tumors. However, it is clear that the extent of apoptosis induced by radiation varies 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 mechanism and arrests the cell cycle at G1 to allow extra time for repair. If repair fails, p53 may trigger deletion of the cell by apoptosis. Cogent evidence for involvement of the p53 gene in the induction of apoptosis by radiation has been provided by the discovery that thymocytes lacking p53 are resistant to the lethal effects of radiation but retain their normal propensity to undergo apoptosis after treatment with glucocorticoids.152~153 However, it should be noted that the last step in the sequence proposed, induction of apoptosis by an increase in the level of the normal (wild-type) p53 gene product, appears to have been demonstrated only in tumor-derived cell lines. 28*36

Induction of Apoptosis by Cancer Chemotherapeutic Agents

A variety of anti-cancer drugs have been shown to induce extensive apoptosis in rapidly proliferating normal cell populations, lymphoid tissues, and tumors.'-

Thus, enhanced apoptosis is responsible for many of the adverse effects of chemotherapy and for tumor regression.

The way in which anti-cancer drugs induce apoptosis is unknown.155 Better understanding of the processes involved clearly might be expected to lead to improved treatment regimen^.'^' However, there is an additional important consequence of the realization that

15,116,154


anti-cancer drugs mediate their therapeutic effect by triggering apoptosis. As has been stressed, apoptosis is a regulated phenomenon capable of being inhibited and activated. Herein may lie a novel explanation for certain instances of drug resistance. Indeed, there is evidence that stimulation of some cell lines by trophic cytokines or increase in their level of expression of the bcl-2 proto-oncogene (the bcl-2 gene product inhibits apoptosis occurring in a variety of circumstances; see section on genetic regulation) can greatly increase their resistance to the apoptosis-inducing effect of 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 similar to that described previously for radiation and anti- cancer drugs-rapidly proliferating normal cell populations, 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 another.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-dependent organs, such as the p r ~ s t a t e ~ ~ , " ~ and adrenal ~ortex,"~ that follows withdrawal of trophic hormonal stimulation, and as might be expected, it also is enhanced in hormone-dependent tumors after successful ablation the rap^.'^-'^ In contrast, increased levels of glucocorticoid induce apoptosis of thym~cytes,~' and a similar effect is observed with many lymphocytic leukemias and malignant lymphomas.'02,'62

In view of the possible role of increased bcl-2 proto-oncogene expression in the development of resistance of tumors to anti-cancer drugs, it is of great interest that recent reports indicate that it also may be involved in resistance to hormone therapy. Thus, although bcl-2 expression was found to be virtually undetectable by immunohistochemistry in 13 of 19 cases of androgen-dependent human prostatic cancer, all of the androgen-independent cancers studied, with the exception of tissue obtained from bone marrow 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-lymphocytes and of the cells of a variety of human lymphoid tumor-derived cell lines. The cell membrane antigen to which this antibody attaches was designated APO-1 .164 The Fas antigen, defined by a second monoclonal antibody 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 regression being accompanied by greatly enhanced apoptosis 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 therapeutic 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-mediated 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 inhibitors of protein synthesis43 or by bcl-2 expre~s ion .~~ Dis- tinctive activation mechanisms probably are involved. 175

Genetic Regulation of Apoptosis

In this article we have referred to the regulation of apoptosis by certain proto-oncogenes and the p53 tu-

Apoptosis in Cancer/Kerr et al. 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 apoptosis might share common signal pathways.

More recently it has been shown that antisense oli- gonucleotides corresponding to c-myc block activation-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 regulation 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-oncogene, 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-oncogene 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 proliferation. However, it does not inhibit apoptosis occurring in all circumstances; as has been mentioned, it fails to block apoptosis induced by cytotoxic T-lymphocyte~.~~

The topographic distribution of bcl-2 expression in normal tissues suggests that it plays vital roles in a variety of physiologic processes in which differential cell survival is imp~rtant.~ As far as oncogenesis is concerned, in addition to the synergy with c-myc mentioned, deregulation of bcl-2 expression may contribute to the accumulation of oncogenic mutations by suppressing the apoptotic deletion of cells that normally follows the induction of DNA damage by agents such as rad ia t i~n . ~~

Enhancement of Apoptosis After Znduced Expression of the p53 Tumor Suppressor Gene

Abnormalities of the p53 tumor suppressor gene, ranging 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 proliferation. By introducing the wild-type p53 gene into cell lines lacking normal p53 activity, a number of p53-mediated 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, respectively, have been found to respond to induced expression of wild-type p53 with the extensive occurrence of apoptos i~ . ~*~~

To what extent p53 is involved in regulating apoptosis 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 subject to stimulation and inhibition. Although little is


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

References

1. Raff MC. Social controls on cell survival and cell death. Nature

2. Williams GT. Programmed cell death: apoptosis and oncogene-

3. Marx J. Cell death studies yield cancer clues. Science 1993;

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 research. 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 electron-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

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

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.

1992; 356:397-400.

sis. Cell 1991; 65:1097-8.

259:760-1.

1986; 19:311-24.

1991; 641192-6.

15. Johnston JB, Lee K, Verburg L, Blondal J, Mowat MRA, Israels LG, et al. Induction of apoptosis in CD4+ prolymphocytic leukemia by deoxyadenosine and 2-deoxycoformycin. Leuk Res 1992;

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

17. Harmon BV, Takano YS, Winterford CM, Gob6 GC. The role of apoptosis in the response of cells and tumours to mild hyperthermia. In t ] 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

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 proliferation and enhance apoptotic cell death of human prostate carcinoma (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 expression. Br I Cancer 1987; 56:251-9.

23. Buttyan R, Zakeri Z, Lockshin R, Wolgemuth D. Cascade induction of c-fos, c-myc, and heat shock 70K transcripts during regression of the rat ventral prostate gland. Mol Endocrinol 1988;

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

27. Hockenbery DM, Zutter M, Hickey W, Nahm M, Korsmeyer SJ. BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci USA 1991;

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 human 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;

31. Colotta F, Polentarutti N, Sironi M, Mantovani A. Expression and involvement of c-fos and c-jun protooncogenes in programmed 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

16:781-8.

1990; 58:845-58.

1991; 163~329-36.

21650-7.

1991; 6:1915-22.

88:6961-5.

359:552-4.

Apoptosis in Cancer/Kerr e t al. 2023

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 between c-myc and bcl-2 proto-oncogenes. Natzrre 1992; 359:

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 resistance 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 factor-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 associated with endogenous endonuclease activation. Nature

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;

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~iol Rev 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 normal 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, 1 9 8 7: 9 3- 1 2 8.

54. Arends MJ, Wyllie AH. Apoptosis: mechanisms and roles in pathology. 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

554-6.

1980; 284~555-6.

7~115-9.

normal liver and in altered hepatic foci of rats. Carcinogenesis

56. Kyprianou N, Isaacs JT. Activation of programmed cell death in the rat ventral prostate after castration. Eiidocrinology 1988;

57. Rotello RJ, Hocker MB, Gerschenson LE. Biochemical evidence for programmed cell death in rabbit uterine epithelium. A m 1

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;

59. Yanagihara K, Tsumuraya M. Transforming growth factor & induces apoptotic cell death in cultured human gastric carcinoma 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 absence of internucleosomal DNA fragmentation. Biochem 1992;

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-dependent 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 glucocorticoid-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 characterization of a calcium-dependent endonuclease (NUCIS) from apoptotic rat thymocytes: NUC18 is not histone H,B. Biol Chem

69. Barry MA, Eastman A. Identification of deoxyribonuclease I1 as an endonuclease involved in apoptosis. Arch Biochem Biophys

70. Peitsch MC, Polzar B, Stephan H, Crompton T, MacDonald HR, Mannherz HG, et al. Characterization of the endogenous deoxyribonuclease 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: purification and properties of thymocytes undergoing apoptosis after glucocorticoid treatment. A m ] Pathol 1982; 109:78-87.

1990; 11:847-53.

122:552-62.

Path02 1989; 134:491-5.

146: 1072-6.

2861331-4.

1991; 266: 18580-5.

1993; 300:440-50.


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 (B id ) 1976; 192241-55.

76. Matter A. Microcinematographic and electron microscopic analysis of target cell lysis induced by cytotoxic T lymphocytes. Immu- l~ology 1979; 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, 1993:3 15-23.

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

80. Fesus L, Thomazy V. Searching for the function of tissue transglutaminase: its possible involvement in the biochemical pathway of programmed cell death. Adv Exp Med Biol 1988;

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 (apoptosis). 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-

87. Savill J, Dransfield I, Hogg N, Haslett C. Vitronectin receptor- mediated phagocytosis of cells undergoing apoptosis. Nature

88. Savill J, Hogg N, Ren Y, Haslett C. Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J Clin Invest 1992;

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 programmed 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.

1987; 224:104-8.

231 :I 19-34.

O ~ Y 1985; 56:351-8.

1990; 343:170-3.

90: 15 13-22.

92. Owens GP, Hahn WE, Cohen JJ. Identification of mRNAs associated with programmed cell death in immature thymocytes. Mol Cell Biol 1991; 11:4177-88.

93. Owens GP, Cohen JJ. Identification of genes involved in programmed 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 superfamily, upon programmed cell death. EMBO J 1992; 11:3887-95.

95. Wyllie AH, Morris RG, Smith AL, Dunlop D. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol

96. Sellins KS, Cohen JJ. Gene induction by y-irradiation leads to DNA fragmentation in lymphocytes. J Znimunol 1987;

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 apoptosis. 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;

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;

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-dioxin kills immature thymocytes by Ca2+-mediated endonuclease activation. Science 1988;

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;

105. Ferrer I. The effect of cycloheximide on natural and x-ray-induced cell death in the developing cerebral cortex. Brain Res 1992; 588:351-7.

106. McConkey DJ, Orrenius S, Jondal M. Cellular signalling in programmed 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;

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 metamorphosis with special reference to apoptosis of striated muscle fibres. J Cell Sci 1974; 14:571-85.

11 1 . Kerr JFR. Shrinkage necrosis: a distinct mode of cellular death. J Pathol 1971; 105:13-20.

1984; 142~67-77.

139~3 199-206.

145: 1859-67.

64: 518-22.

242:256-9.

143~3461-9.

87:465-71.

Apoptosis in Cancer/Kerr et al. 2025

112. Benedetti A, JGzGquel AM, 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 castration-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

118. Duke RC, Cohen JJ. IL-2 addiction: withdrawal of growth factor activates a suicide program in dependent T cells. Lymphokine Res

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-dependent hemopoietic cells. ] Immunol 1992; 149:535-40.

123. Lynch MP, Nawaz S, Gerschenson LE. Evidence for soluble factors regulating cell death and cell proliferation in primary cultures 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

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 apoptosis (programmed cell death) in granulosa cells: evidence for a potential mechanism underlying follicular atresia. Eiidocriiiology

127. Weedon D, Strutton G. Apoptosis as the mechanism of the involution 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;

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 inflammation: 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 occurrence of apoptosis in squamous carcinomas of the uterine cervix.

Atiat 1979; 156:15-36.

1986; 5:289-99.

1989; 185:19-32.

1991; 129:2415-22.

337~181-4.

Pathology 1973; 5:163-9.

133. Wyllie AH. The biology of cell death in tumours. Aitticaiicer Res

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

136. Gob6 GC, Axelsen RA, Searle JW. Cellular events in experimental 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;

138. Wright SC, Kumar P, Tam AW, Shen N, Varma M, Larrick JW. Apoptosis and DNA fragmentation precede TNF-induced cytolysis in U937 cells. ]Cell Biochem 1992; 48:344-55.

139. Curson C, Weedon D. Spontaneous regression in basal cell carcinomas. 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 preneoplastic and neoplastic liver cells: a sequential study. A m ] Pathol

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 injury 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

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 32P induces massive apoptosis in a Burkitts lymphoma cell line. lnt ] Radiat Biol

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-dependent 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-

1985; 5~131-6.

1988; 21:45-9.

52~1342-6.

1984; 116:441-6.

1989; 120: 140-53.

1992; 61~365-7.


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 induced by transforming growth factor pl and cancer chemotherapy compounds in myeloid leukemic cells. Blood 1992; 80:

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 hyperthermia 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 thymocytes. 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-independent 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 induction 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 human cell surface antigen Fas can mediate apoptosis. Cell 1991;

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 antigen. ] Biol Chem 1992; 267:10709-15.

1750-7.

66~233-43.

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 specifically allergized lymphocytes in vitro. Aus t Exp Biol Med Sci

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

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;

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

1977; 55:407-17.

SOC Lor~d (Biol) 1977; 197~417-24.

6:3 169-76.

1992; 356:2 15-21. 179. Lane DP. A death in the life of p53. Nature 1993; 362786-7.

Date post:	11-Sep-2015
Category:	Documents
Upload:	rafael-archila
View:	215 times
Download:	0 times

Apoptosis John F. R. Kerr, Ph.D.,* Clay M. Winterford

Documents