Programmed cell death: concept, mechanism and control.

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Biol. Rev. (1992), 67, p p . 287-319 Printed in Great Britain

PROGRAMMED CELL DEATH: CONCEPT, MECHANISM AND CONTROL BY SOUMITRA SEN” Centre for Advanced Study in Cell and Chromosome Research, University of Calcutta, 3 5 Ballygunge Circular Road, Calcutta 700 0 19, India (Received

10 October

1991 ; revised 19 February 1992 ; accepted 30 April 1992)

Dedicated to Professor S. Garattini, Director and to Professor A. Leonardi, Secretary General and Dean, Istituto di Ricerche Farmacologiche ‘Mario Negri ’, Milan, with respect and gratitude. CONTENTS I. Introduction . . . . . . . . . . . . . . . . ( I ) Types of cell death . . . . . 11. Concept of programmed cell death . . . ( I ) Early evidences and genesis of the concept (2) Determination of the death process . . . . 111. Mechanism of apoptosis . . . . . . . ( I ) Initiating stimuli . . . . . . . . (2) Effector mechanisms . . . . . . . . . ( a ) Precommitment and commitment steps . (3) Morphological changes . . . . . . . . . . . . ( a ) Reduction of cell volume . (b) Changes in the nucleus . . . . . . (c) Changes in plasma membrane . . . . . (4) Biochemical events . . . . . . . ( a ) Endonuclease activation . . . . . . (b) Role of calcium . . . . . . . ( c ) Receptors and signal transduction . . . . (d) Macromolecular synthesis . . . . . . ( e ) Gene activation . . . . . . . IV. Influence of cell cycle phase and possible molecular regulation V. Significance of apoptosis . . . . . . . VI. Summary and conclusions . . . . . . . VII. Acknowledgements . . . . . . . . VIII. References . . . . . . . . .

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I. INTRODUCTION

Cell death is an essential strategy for control of dynamic balance of the living system. It is the ultimate result of most pathological processes. Like most physiological events, death too is initiated through the perception of specific and defined stimuli. This leads to a cascade of intracellular changes and culminates in subsequent effector events. ( I ) Types of cell death

Studies on dying cells reveal two definite patterns.

* Present address for all correspondence: Istituto di Ricerche Farmacologiche ‘Mario Negri’, via Eritrea 62, 1-20157 Milan, Italy.

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( a ) Traumatic or necrotic death: where the cell suddenly confronted with extreme non-physiological conditions loses control of ion-flux. In the second step, calcium enters the mitochondria ; continued metabolism makes the cell hypertonic to its milieu, water flows in and the cell together with its organelles swell and lyse (Trump, Berezesky & Osornio-Vargas, I 98 I ) . Uncontrolled swelling and dilatation of mitochondria are typical features of this kind of cell death. ( b ) Apoptotic or programmed cell death: occurs in physiological conditions, often as a result of physiological effectors which are not lethal to other cells in the vicinity. It is a relatively slower process than necrosis (Kyprianou & Isaacs, 1989) and involves a series of well-regulated synthetic events. During apoptosis, the dying cell induces a ‘programme’ so that a series of degenerative changes occur in response to the effector stimuli. T h e cell actively participates in this ‘suicidal ’ process and destroys itself. This kind of death was originally recognized by Kerr, Wyllie & Currie (1972) who coined the term ‘ apoptosis ’ to specifically distinguish programmed cell death from necrotic death (Kerr, 1971; Kerr et a l . , 1972; Wyllie, Kerr & Currie, 1980; Wyllie, 1987). Interest in the field of programmed cell death produced a wealth of information in recent years. T h e present review would be concentrated on the determination of death processes, effector mechanisms, morphological and biochemical changes during programmed death and on its possible molecular regulation. 11. CONCEPT OF PROGRAMMED CELL DEATH

Physiological cell death has been recognized since the early days of embryology but was first emphasized by Glucksmann ( I 95 I ) and subsequently re-recognized in 1960s. Experiments demonstrated that embryonic cells or metamorphosing insect cells carried within themselves the information for their own destruction at a particular time. It was postulated that these cells contained a programme or biological clock set for death well prior to the time the programme was actually executed.

( I ) Early evidence and genesis of the concept During development, programmed cell death occurred in a wide range of circumstances. T h e classical examples were cited by Saunders, Gasseling & Saunders (1962); Lockshin & Williams (1964) and by Saunders (1966) to describe development (see, Lockshin & Beaulaton, 1974; Beaulaton & Lockshin, 1982; Umansky, 1982). It was shown that the signal was a chemical (mostly a hormone or a growth factor) and was not toxic to other cells (Lockshin, 1981).Under experimental conditions, cells could be prevented from dying in presence of the signal. Beaulaton & Lockshin, (1978) had shown that where the targeted cells were situated among other cells, the general mass of cells remained non-responding to and unaffected by the signal. In metamorphosing moths, death of specific larval or pupal neurones occurred in specific sequence - each cell followed its own timing and sequence of death (Schwartz & Truman, 1982; Truman & Schwartz, 1982a, b ; Truman, 1983 ; Lockshin & Zakeri-Milovanovic, I 984). Similar observations were also reported about ecdysterone and eclosion-induced programmed muscular degeneration in the giant silkmoth (Lockshin, 1971 ; Truman & Schwartz, 1980) and hawkmoth (Truman & Schwartz, 1982a, 6 ; Truman, 1983). In vertebrates, specific muscle involved in sexual activity are responsive to androgens (Hanzlikova & Gutmann, I 974, I 978). Hormone-dependent apoptosis have been

Programmed cell death Table

I.

289

Examples of physical and chemical agents inducing apoptotic death

Agent Physical agents Cold Heat

Ultraviolet light X-rays P-rays y-rays Neutrons Virus HIV-I Trace metal Zinc Hormones/growth factors Fibroblast growth factor Deprivation Transforming growth factor I Interleukin-3 Serum deprivation Glucocorticoids

1-

Diethylstilbestrol Transforming growth factor PI Antibodies Anti-immunoglobulin Anti-APO- I

Antibody Anti-CDq+CDV Anti-CDz3 Anti-CD77 Anti-IgM Monoclonal antibodies

Test system

Reference

Chinese hamster V79 cells Chinese hamster cells Lymphocytes Lymphoblastoid line Thymocytes Mastocytoma Chinese hamster ovary (CHO) cells Human and murine tumour cell lines Molt-4, U937, HL60, Daudi Guinea pig skin HL60 Rat thymus Mouse intestinal cells Lymphocytes Human peripheral leukocytes Mouse small intestinal cells

Soloff et al. (1987) Nagle et a1 (1990) Shrek et al. (1980) Dyson et al. (1986) Sellins & Cohen (1991) Harmon et al. (1990) Barry et al. (1990) Takano et al. (1991) Lennon et al. (1991) Danno & Horio (1982) Martin & Cotter (1991) Ohyama et al. (1985) Ijiri, 1989 Sellins & Cohen (1987) Tomei et al. (1990) Hendry & Potten (1982) Hendry et al. (1982)

T lymphoblasts

Terai et al. (1991)

Pancreas

Kazacos & Van Vleet (1989)

Endothelial cells

Araki et al. (1990)

Uterine epithelial cells Haemopoitic cells Lymphocytes Thymocytes Neoplastic (lymphoid) cells Mouse P1798 lymphoma cells CEM C I , CEM C7 cells Chronic lymphocytic leukemia H3o I estrogen-dependent kidney tumour in vivo

Rotello et al. (1991) Rodriguez-Tarduchy, et al. (1990) Lucas et al. (1991) Wyllie (1980) Bansal et al. (1990) Thompson (1991) Bansal et al. (1991) Forbes et al. (1992) Bursch et al. (1991)

Human hepatoma Hep 3B

Lin & Chou (1992)

WEHI 23 I B lymphoma Activated human lymphocytes Malignant lymphocytes Leukemic cells from patients Lymphocytes Thymocytes Germinal centre B cells

Benhamou et al. (1990) Trauth et al. (1989) Trauth et al., (1989) Trauth et al. (1989) Stacey et al. (1985) Murphy et al. ( I 990) Liu et al. (1991) Mangeney et al. (1991) Ishigami et al. (1992) Janssen et al. (1991) Janssen et al. (1991) MacDonald & Lees (1990)

Human B104, WEHI-231 Thymocytes IL-?-dependent gd T cells Autoreactive thymocytes

+

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290 Agent

Test system

Foetal mouse thymic organ culture Anticancer chemotherapeutic agents Thymocytes Amsacrine CHO strain AA8 Aphidicolin CHO strains AA8, UV41 I -p-D-arabinofuraHL60, K G I A nosyicytosine CCRF/CEM C 7 , BCNU F89, Molt-4-F, E B I , EBz-3945 Burkitt’s lymphoma lines bis-(2-chloroethy1)methylamine HL60, K G I A Camptothecin CHO strains AA8, UV41 Cisplatin HL60, K G I A

L I 2 1010 Etoposide

5-fluorodeoxyuridine 5-fluorouracil

llethotrexate Melphalan

Teniposide Vincristine

Others TNF

Gliotoxin Sporidesmin N K cells Cytotoxic T cells Cytroterone acetate Methylprednesolone Phenobarbital promotion I ,z-dimethylhydrazine Methylazoxymethanol TPA DHTB Isoproterenol Retinoic acid Calcium ionophore

Thymocytes CHO strains AA8, UV4i HL60, K G I A CHO Chronic lymphocytic leukemia C H O strains AA8, UV41 C H O strains AA8, UV4i CCRF/CEM C7, F89, Molt-4-F, E B i , EBz-3945 C H O strains AA8, UV41 HL60, K G i A CCRF/CEM C7, F89, Molt-+F, EBI, EBZ-3945 Thymocytes C H O strain AA8 F89, Molt-4-F, E B I , EBz-3945

Reference Smith et al. (1989)

Walker et a / . (1991) Kung et al. (1990) Barry et al. (1990) Kaufmann (1989) Dyson et al.

( I 986)

O’Connor et al. (1991) Kaufmann ( I 989) Barry et a/.(1990) Kaufmann (1989) Sorenson et al. (1990) Walker et al. (1991) Barry et al. ( I 990) Kaufmann (1989) Lock & Ross ( 1 9 9 0 ~ ) Forbes et al. (1992) Barry et al. (1990) Barry et al. (1990) Dyson et al. (1986)

Barry et al. (1990) Kaufmann ( I 989) Dyson et al. ( I 986)

Walker et al. (1991) Kung et al. (1990) Dyson et al. (1986)

Macrophages, T blasts LAK cells Murine and human cells Rat liver cells Lymphoblastoid cell line Rat liver

Laster et al. (1988) Robaye et al. (1991) Rodriguez-Tarduchy et al. (1990) Waring et al. (1988) Waring (1990) Waring et al. (1990) Liu et al. (1989) Zychlinsky et a / . (1991) Bursch et al. (1984) Dyson et al. (1986) Schulte-Hermann el al. (1990)

Mouse small intestine Mouse small intestine Z C3H I O T I / cells cells C3H IOTI/P Thymocytes HL60 cells HL60 cells

Kenichi (1989) Kenichi (1989) Tomei et al. (1981) Tomei et al. (1981) Tadakuma & Kizaki (1991) Martin et al. (1991) Martin et al. (1990)

Endothelial cells Haemopoitic cells Macrophages

Programmed cell death Agent

Microtubule poison Okadaic acid

Tributyltin Methanol Dimethylsulphoxide Sodium azide Ethanol Hydrogen peroxide Chlorambucil Amethopterin Acetaminophen

291

Test system

Reference

HL60 cells Lymphocytes HL60 cells Chronic lymphocytic leukemia Rat pituitary adenoma cell line Rat primary hepatocytes MCF 7; SK-N-SH, IPC 81 ; GH3 cell lines Rat thymocytes HL60 cells HL60 cells HL6o cells HL60; Molt-4; U937, Daudi HL60; Molt-4; U937, Daudi HL60; Molt-4; U937, Daudi HL60; Molt-4; U937, Daudi Mouse hepatocytes in vitro

Lennon et al. (1991) Lucas et al. (1991) Martin & Cotter (1989) Forbes et al. (1992) Boe et al. (1991) Boe et al. (1991) Boe et al. (1991) Aw et al. (1990) Lennon et al. (1991) Lennon et al. (1991) Lennon et al. (1991) Lennon et al. (1991) Lennon et al. (1991) Lennon et al. (1991) Lennon et al. (1991) Shen et al. (1991)

reported in adrenal cortex following withdrawal of adrenocorticotropic hormone (Wyllie et al., 1973a , b), in the prostate in absence of testosterone (Kerr & Searle, I973), in the regressing corpora lutea (O’Shea, Hay & Cran, 1978), in the endometrium (Hopwood & Levison, 1976; Sandow et al., 1979), and in the breast epithelium (Ferguson & Anderson, 1981a , 6). Genetic lesions in avian mutants produce either extremely high or low levels of apoptosis during development of limb (Hinchliffe & Ede, 1973; Hinchliffe & Thorogood, 1974) and indicated a genetic control over the process. A classical example of apoptotic death giving rise to the concept of biological death-clock comes from the study of regulation of cell death of the posterior necrotic zone of chicken wing. These cells die in stage 24 of embryonic development. Until this time it is impossible to distinguish them from their neighbours. When grafted to other sites or explanted in culture, these cells show apoptotic changes and eventually die at the time corresponding to stage 24 (Fallon & Saunders, 1968) despite the change in their position or environment. These early observations helped to synthesise the concept that physiological cell death is controlled by precise intrinsic genetic programme. The cells ‘ remember ’ the programme while the initiating stimulus can result in cell to cell interactions. Apoptotic cell death requires intracellular metabolism and active participation of the dying cells. (2)

Determination of the death process

The stimuli that initiate death vary widely with the affected cells [see Section I I I . ( I ) ] . Apart from hormones and growth factors, apoptosis may be induced by low levels of y-irradiation (Sellins & Cohen, 1987), lymphotoxin (Schmid, Tite & Ruddle, 1986), tumour necrosis factor and related cytotoxins (Liu et al., 1987; Laster, Wood & Gooding, 1988), several non-physiological toxins (McConkey et al., 1988) and even by extracellular ATP (Zheng et al., 1991). Apoptosis can be induced after minor injury caused by low doses of ionizing irradiation. Such treatment causes single strand breaks in DNA of cortical thymocytes.

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T h e damage is rapidly repaired (Scaife, 1972; Filippovich et a l . , 1982). T h e apoptotic death process is triggered on after repair of the damage in physiologically active cells (Skalka, Matyasova & Cejkova, 1976; Yamada et al., 1981). Potten, Al-Barwari & Searle (1978) and Potten et al. (1983) demonstrated that apoptosis can also be triggered by injured cells. When a sub-population of extra-sensitive cells are damaged, apoptosis results due to interaction between the sensitive cells and their surrounding cells. Apoptosis may be induced by low concentration or level of almost all those stimuli that cause necrosis (Lennon, Martin & Cotter, 1991). T h i s means that the mechanism of self-destruction can be activated by a relatively mild stimulus. While mild hypoxia produced symptoms of apoptosis, severe hypoxia produced infarction and necrosis (Kerr, 1971). While exposure to temperatures between 37-43 "C induced apoptosis, exposure to higher temperatures induced necrosis in lymphocytes (Shrek et al., 1980). Cultured Chinese hamster cells undergo apoptosis even after brief exposure to cold (0-6 " C ) (Soloff et a l . , 1987). Recently it has been shown that in murine mastocytoma cells, temperature clearly determines the mode of death. T h e form of death changes from apoptosis to necrosis above a critical heat load (Harmon et al., 1990). It is suggested that cells possess effector mechanisms for self destruction which may be activated by several mechanisms and by various relatively mild stimuli. Unfortunately we understand very little about the perception of the stimulus, the role of the intensity or severity of the stimulus and the actual process that determines the mode of cell death. 111. MECHANISM OF APOPTOSIS

( I ) Initiating stimuli

A tremendous variety of both physical and chemical stimuli can induce apoptosis. Table I summarizes information available on the varied nature of initiating stimuli. (2)

Effector mechanisms

T h e initial effector in response to the stimulus has been little studied and very poorly understood. It seems to be a signal that is responsible for a sudden rise in intracellular Ca2+ level. T h i s is discussed in detail in Section III(46). Whatever be the primary effector, a cell destined to die exhibits at least two major steps: pre-commitment and commitment steps.

( a ) Pre-commitment and commitment steps Cell killing by cytotoxic T cells in vitro (Sanderson, 1976, 1981; Matter, 1979; Russell & Dobes, 1980; Duke, Chervenak & Cohen, 1983; Russell, 1984; Tirosh & Barke, 1985; Martz & Howell, 1989; Zychlinsky et a l . , 1990; Liu, Mullbacher & Waring, 1989) demonstrated three phases in the apoptotic death process: ( I ) recognition, (2) pre-commitment and (3) commitment steps. T h e recognition involves binding or adherence of the cytotoxic T cells to the target cells and is mediated through receptors (Meuer et a l . , 1985). During pre-commitment step, the cytotoxic T cell passes its pseudopodia within the target cell and the latter becomes metabolically active. Temperature plays a very crucial role - the programming is not complete if the temperature of the target cell is below the physiological temperature (Martz, Burakoff & Benacerraf, 1974). T h e commitment step is

Programmed cell death

293

independent of the presence of the T cell. During this phase the cells show activation of the endonuclease. Once the cytotoxic T cell extends its pseudopodia into the target cell, the killing could not be inhibited by inhibitors of protein synthesis (Duke et al., I 983). Interestingly, when the target cells, pre-treated with the macromolecular synthesis inhibitors, are incubated with lymphokine-activated killer cells, showed morphologic signs of necrosis and not that of apoptosis (Zychlinsky et al., 1991).This indicates that a pre-commitment step for induction of genetic programmation through macromolecular synthesis is essential for death to occur through apoptotic cascade. Studies with the inhibitors of protein and RNA synthesis on y-irradiated lymphocytes clearly showed that once the apoptotic cascade of metabolic processes has been initiated within the individual cell, it proceeds to completion (Sellins & Cohen, 1987).Furthermore, the persisting inducing signal is not simply mRNA molecules, but an active genetic programme persisting within the committed cell (even in presence of inhibitors) that induces the cell to enter a suicidal mode of destruction upon removal of the inhibitor. ( 3 ) Morphological changes Apoptotic cells undergo a transient but distinct increase in bouyant density (Ohyama, Yamada & Watanabe, 1981;Thomas & Bell, 1981;Wyllie & Morris, 1982). This permitted harvest of purified apoptotic cells. Morphological changes have been described in great details by Wyllie et al. (1980)and by Wyllie (1981). ( a ) Reduction in volume

Water is lost from the cells without loss of macromolecules. Cells at this stage do not show increased membrane permeability (measured in terms of vital dye uptake or radioactive chromium release). Reduction of volume is an outstanding feature of apoptotic cells (Kerr, 1971).Shrinkage corresponds well with the onset of programmed cell death determined biochemically (Sorensen, Barry & Eastman, 1990). Apoptotic cells lose contact with neighbouring cells and subsequently show chromatin condensation and compaction of cytoplasmic organelles. At a later stage, these appear as membrane-bound fragments known as apoptotic bodies (Kerr et al., 1972; Wyllie et al., 1980).

( b ) Changes in the nucleus Chromatin gradually becomes granular, intensely osmiophilic in character ; extending as a band underneath the inner lamina of the nuclear membrane. Prominent nuclear pores appear as patches. Major part of the periphery of the nucleus is occupied by condensed chromatin. Peripheral chromatin of the nucleolus disintegrates as small osmiophilic granules. Parts of the chromatin exhibit argyrophilia and remain less osmiophilic -these have been identified as protein-rich zones (Wyllie et al., 1980; Wyllie & Morris, 1982).Ultimately the nucleus is represented by condensed granular scattered fragments. The nuclear membrane might exhibit convolutions and may still maintain its dual membrane structure with pores. Chromatin of eukaryotic cells is anchored to sites on the protein matrix of the nucleus. In thymocytes, such anchorage points have been identified to be associated with the nuclear envelope (Cavazza et a l . , 1983).Treatment of the apoptotic thymocyte nuclei with high concentration of salt or mineral acid showed that the protein sub-

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structure of the nucleus is initially retained together with the anchored DNA. The nuclease cleavage between the anchorage points destroys the integrity of the looped domains at a later stage (Wyllie et al., 1984b).

( c ) Changes in plasma membrane Both morphological and chemical changes in the plasma membrane of the apoptotic cells permit their recognition by adjacent cells and by phagocytes. T h e process of recognition has been shown to be fundamental in control of homeostasis (Hedgecock, Sulston & Thomson, 1983). Apoptotic thymocyte is an ideal model (Morris et al., 1984) since highly purified cell populations can be obtained easily. T h e surface charge density of apoptotic cells is slightly less. Studies with neuraminidase indicated that that terminal N-acetyl neuraminic acid (NANA) groups are lacking in the plasma membrane. Selective removal of NANA from pre-existing glycan chains or incomplete synthesis of the chains could be responsible. Alterations in significant proportion of surface carbohydrates take place that help in preferential binding of macrophages to apoptotic cells (Duvall, Wyllie & Morris, 1985). Data suggests that presence of lectin-like molecules on the surface of macrophages recognise changes in cell surface carbohydrates. Binding of the macrophages in inhibited by N acetyl glucosamine and its dimer, N,N,-diacetyl chitobiose (Wyllie, Duvall & Blow, 1 9 8 4 ~ )Recognition . of apoptotic cells by macrophage has been shown to be mediated by the vitronectin receptor, a heterodimer belonging to the cytoadhesin family of the integrins. Anti-vitronectin receptor antibody, 23C6, inhibited the interaction by over 7 o o o (Savill et al., 1990) indicating a direct role of the vitronectin receptors in recognition process. Dilated endoplasmic reticulums fuse and burst with plasma membrane (Yamada & Ohyama, 1 9 8 0 ; Galili et al., Morris et al., 1984). T h e plasma membrane loses its characteristic structure and projections and show blebbing. It also buds off projections so that the whole dying cell may split into membrane-bound apoptotic bodies.

( 4 ) Biochemical eaents T h e most detailed studies on the mechanism and biochemical events were performed on immature thymocytes. T h e following is the summary of biochemical events recorded in apoptotic cell systems.

( a ) Endonuclease activation T h e double-stranded linker D N A between nucleosomes is cleaved at regularly spaced inter-nucleosomal sites, giving rise to D N A fragments the length of which represent the length of nucleosomes ( I 80-200 base pairs). This typical cleavage is due to activation of an endogenous endonuclease (Kerr, 1 9 7 3 ; Skalka et al., 1 9 7 6 ; Appleby & Modak, 1 9 7 7 ; Wyllie, 1980, 1 9 8 1 ;Wyllie et al., 1 9 8 0 ; Yamada et al., 1981; Wyllie & Morris, 1 9 8 2 ; Manes & Menzen, 1982). Cleavage of DNA generates two classes of chromatin fragments: 7 o 0 0 of the D N A exists as long HI-rich oligonucleosomes bound to the nucleus and 30 'lo comprising of short oligonucleosome and mononucleosome which are depleted in HI, and enriched in (high mobility group proteins) H M G , and HMG, and not attached to the nucleus. This typical chromatin cleavage, nucleolar morphologic changes and chromatin condensation were closely mimicked by micrococcal nuclease


295

digestion of normal thymocyte nuclei in the presence of protease inhibitors. This implied selective activation of an endogenous endonuclease is responsible for the cleavage. During the endonuclease activity, proteases remain inactive (Arends, Morris & Wyllie, 1990). Just the contrary chromatin changes were observed in case of necrosis (Russell, 1984). The specific endonuclease is yet to be identified. But the nature of the enzyme is known. It is a calcium-magnesium-sensitive neutral endonuclease, known to be present in lymphocytes (Kaiser & Edelman, 1977) and in many terminally differentiated cells (Wyllie, 1980; Nakamura et al., 1981; Cohen & Duke, 1984). Inhibitors of protein synthesis caused a fall in the enzyme level (Wyllie et al., 19846). Zinc is a potent inhibitor of the enzyme (Duke et al., 1983 ; Cohen & Duke, 1984). Concentration of zinc ion is higher in cell nucleus under normal physiologic conditions (Wester, 1965 ; Larsen et al., 1982). Acute rise in the programmed death in lymphoid and myeloid cells lines during zinc deficiency has recently been reported (Martin et al., 1991). It is possible that the endonuclease is activated by an exchange of calcium and magnesium for zinc in the nucleus. Support of this view comes from the observation that zinc deficiency causes thymic involution in vivo (Fraker, Haas & Leucke, 1977; Fernandes et al., 1979); enhances killing of gastrointestinal epithelium (Elmes, 1977 ; Elmes & Gwyn Jones, 1980) and of developing Mullerian duct tissues in response to Mullerian inhibitory substance (Budzic et al., 1982). Zinc renders protection against apoptosis induced by the mycotoxin, sporidesmin, in macrophages, T blasts (Waring et al., 1990) and in thymocytes (Cohen & Duke, 1984). Spermine was active against glucocorticoid- and Ca2+ionophore-induced apoptosis by intercalating with the endonuclease or its substrate DNA. Thymocytes, when incubated with methylglyoxal bis (guanylhydrazone) to deplete intracellular spermine, showed spontaneous DNA fragmentation typical of apoptosis (Briine et al., 1991). The increased endonuclease activity could be the result of either activation of the protein through increased intracellular calcium (McConkey et al., 19896) or through induction of new proteins having endonuclease activity, similar to that described by Compton & Cidlowski (1987). The existence of the endonuclease was subsequently challenged (Alnemri & Litwack, 1989; Baxter, Smith & Lavin, 1989; Van den Bogert et al., 1989) based on the fact that histones can express nuclease activity in nuclease gels due to their ability to block intercalation of ethydium bromide into DNA. Very recently, Gaido & Cidlowski (1991) purified and characterized the nuclease from apoptotic rat thymocytes. Their work clearly demonstrated that enzymatically active purified nuclease failed to react with two different anti-histone H,B antibodies. T h e nuclease, NUC 18, originally shown to have a molecular weight of 18 kDa (Compton & Cidlowski, 1987), requires a neutral p H for activity. It is a basic protein with a PI of 8.5 or greater and is calcium dependent. NUC 18 is unrelated to DNaseI and is inhibited by both zinc and aurintricarboxylic acid. The nuclease is present in extracts from both control and dexamethasone-treated thymocytes as a high-molecular-weight ( > IOO kDa) complex. However, the extracts from apoptotic thymocytes contain the nuclease in fractions of gel filtration the molecular weight of which corresponds to about 25 kDa. Since the untreated control cells contain only the high molecular weight form, it is possible that the hormone treatment stimulates the synthesis of N U C 18 or release of the smaller molecular weight form from the high molecular weight

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complex/precursor. T h e authors postulated that it is possible that following dexamethasone treatment, the nuclease is activated by transcriptional activation via the classical steroid receptor pathway. Purification and characterization of the nuclease would provide identification of the specific gene encoding the protein. It might be possible then to selectively modulate the gene action to induce a programmation for death in specific cells.

(6) Role of calcium In thymocytes, increased CAMP concentrations stimulate D N A fragmentation without activation of protein kinase C, deemed to be responsible for increase in Ca2+ concentration (McConkey, Orrenius & Jondal, I 990 a). Recently, involvement of cyclic adenosine monophosphate-kinase I has been shown in apoptotic myeloid leukemia cell line (Lanotte et al., 1991).Apoptosis could be induced by polymixin B, an inhibitor of protein kinase C (Tomei, Kanter & Wenner, 1981; Tomei et al., 1990; Lucas, Solano & Sanz, 1991). Apoptosis was hindered in presence of phorbol myristate acetate (Benhamou, Cazenave & Sarthou, I 990 ; Rodriguez-Tarduchy, Collins & Lopez-Rivas, 1990) and phorbol I 2 , I 3-dibutyrate (Rodriguez-Tarduchy & Lopez-Rivas, I 989). Mobilization of Ca2+ seems to be related to apoptosis induced by gliotoxin. Elevated levels of inositol triphosphate was noted in macrophages treated with gliotoxin (Waring, 1990) and in T lymphocytes grown in absence of interleukin-z (RodriguezTarduchy & Lopez-Rivas, 1989). Inositol triphosphate functions as a second messenger to mobilise Ca2+ from intracellular reservoirs. Recently it has been shown that quintal release may depend on the sensitivity of the inositol triphosphate receptor being regulated by the Ca2' concentration in the lumen of the endoplasmic reticulum (Missiaen, Taylor & Berridge, 199I ) . McConkey et al. ( I 989 a ) reported internucleosomal D N A cleavage in thymocytes treated with calcium ionophore, A23 187. Martikainen et al. (1991) demonstrated that sustained elevation of Ca2+,only 3- to 6-fold above the baseline can induce apoptotic death in androgen-independent Dunning R-3327 rat prostatic cells. Prevention of Ca2+ increase by buffering cytosolic Ca2+ with Quin-z or through incubation in Ca2+-free medium prevented endonuclease activation and cell killing. T h e increase in Ca2+appeared to be due to the action of a heat-labile cytosolic factor, synthetised in response to glucocorticoid treatment (McConkey et al., 1989b ) .

( c ) Receptors and signal transduction Presence of active glucocorticoid receptors is essential for induction of apoptosis by glucocorticoid treatment. T h e receptors have both ligand- and DNA-binding domains. After binding, the receptor-ligand complex migrates into the cell nucleus where it binds to the enhancer sequences known as glucocorticoid receptor enhancer (GRE). T h e biological effects of glucocorticoids are regulated by the gene products whose transcription is controlled by the receptor-GRE interactions. Immature CDq+CD8+ thymocytes are particularly prone to the hormones and serve as a model (Screpanti et al., 1989; Murphy, Heimberger & Loh, 1990). Rescue of thymocytes and T cell hybridomas from glucocorticoid-induced apoptosis by stimulation via the T cell receptor/CD-j complex has been reported (Iwata, Hanaoka & Sato, 1991). Administration of anti-CD3 antibodies produced D N A degradation in via0 (Shi, Sahai &


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Green, 1989; Shi et al., 1991) and in vitro (Stacey et al., 1985; Smith et al., 1989; McConkey et al., i989a). Apoptosis is triggered if a T cell that has received the signal through the T cell receptor complex, also received the signal through the a,-domain of the class I major histocompatibility complex molecule. Such a signal may be delivered by a CD8 molecule that recognizes the a, domain or by an antibody to this domain. Precursors of both CTLs and T helper cells are sensitive to this signal. Because CTLs carry CD8, they can induce death in T cells that recognize them (Sambhara & Miller, 1991). Phorbol esters prevented apoptosis in response to anti-CD3 suggesting that activation of protein kinase C abrogated cell suicide (McConkey et al., 1 9 8 9 ~ )Examination . of functions of the accessory molecules, such as CD4+, CD8+, C D N , shows regulatory functions on responses of immature thymocytes through the TCR/CD3 complex. Crosslinking CD4 or CD8 with CD3 strongly enhanced signal transduction via CD3 as assessed by protein tyrosin phosphorylation and Ca2+mobilization. In absence of CD28 stimulation, the enhanced TCR/CD3 signals may lead to apoptosis (Turka et al., 1991; Ju, 1991). Anti-APO-1 induced apoptosis in a variety of cells including activated human lymphocyte lines and patient-derived leukemic cells in vitro and also in vivo in xenotransplanted human B cell tumour-bearing mice (Trauth et al., 1989). Similarly, administration of dexamethasone induced thymic atrophy in mice in vivo (Lundberg, 1991). CEM C I cells that possess functional glucocorticoid receptors, are resistant to cell killing by dexamethasone (Zawydiwski, Harmon & Thompson, I 983). Lovastatin, a competitive inhibitor of H M G CoA-reductase, the rate limiting enzyme in cholesterogenic pathway, inhibited growth of dexamethasone-sensitive CEM C7 cells, but only slightly affected the growth of the resistant line, CEM C I . RU-486, as antiglucocorticoid compound that acts on the glucocorticoid receptor by preventing its translocation from the cytoplasmic compartment to the nuclear compartment, prevented cytotoxicity of dexamethasone in CEM C7 cells ; but were ineffective on lovastatininduced cytotoxicity (Bansal, Houle & Melnykovych, I 989). The killing of the resistant CEM C I cells by various treatments indicated that these cells possess the elements of an apoptotic cascade that does not involve activation through glucocorticoid receptors (Bansal, Houle & Melnykovych, 1991). Furthermore, RU-486 did not inhibit activation-induced cell killing. It neither inhibited translocation of the glucocorticoid receptors, nor prevented receptor translocation induced by dexamethasone : indicating thereby that cell activation and steroid-induced apoptotic pathways are mutually independent (Zacharchuk et al., 1990) and antagonistic (Iseki, Mukai & Iwata, 1991). Bleomycin, an antibiotic known to release nucleosomes from chromatin and chromosomes (Kuo & Hsu, 1978), induced apoptosis (Lucas et al., 1991) in mature lymphocytes known to be insensitive to phorbol esters and calcium ionophore treatment. Polymixin B induced apoptosis in mature lymphocytes. Though the exact mechanism is unknown (Lucas et al., 1991), inhibition of protein kinase C (Naccache, Molski & Sha'afi, 1988) or gene activation (Schwartz, Kosz & Kay, 1990) might be responsible for polymixin B-induced apoptosis. Activation-induced apoptosis has been described in T-cell hybridomas (Shi et al., I 990). Antimembrane immunoglobulin antibodies exert inhibitory effect in immature B lymphocytes. Co-treatment with the protein kinase C-activator, phorbol- I 2-

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myristate- I 3-acetate, prevented apoptosis. This indicated involvement of signal transduction in the process (Benhamou et al., 1990). Activation of signal transduction pathways protects Balb/c-3T3 fibroblasts against death due to serum deprivation (Tamm & Kikuchi, 1991). Also accessory cell-derived T cell growth hormone, interleukin I (IL- I), prevents T cell receptor-mediated thymocyte apoptosis by a mechanism involving activation of protein kinase C (McConkey et al., 1990). Interleukin-a protects T lymphocytes from glucocorticoid-induced apoptosis (Nieto & Lopez-Rivas, 1989). Similarly, I L - I P and tumour necrotic factor cx ( T N F cx) prevented apoptotic death of monocytes (Mangan & Wahl, 1991)while IL-3, IL-5 and granulocyte macrophage colony-stimulating factor (GM-CSF) prevented apoptotic death of eosinophils indicating thereby selective modulation of apoptosis by specific cytokines (Her et al., 199I ) . In presence of exogenous interleukin-a, anti-TCR/CD3 monoclonal antibodies induced apoptosis in cloned yS+ T cells. Similar T C R / C D 3 signalling can induce apoptosis not only in immature CDq+CD8+thymocytes, but also in interleukin 2-dependent normal T cells, T-cell hybridomas, transformed leukemic T-cell lines, autoreactive thymocytes and in foetal mouse thymic organ culture (Merc'ep et al., 1988; Takahashi, Maecker & Levy, 1989; Ucker, Ashwell & Nickas, 1989; Smith et al., 1989; McDonald & Lees, 1990; Janssen et al., 1991). Cyclosporin A is a potent inhibitor of TCR-mediated lymphokine gene transcription, and has also been shown to prevent binding of proteins to enhancer 5' element of the interleukin-a gene (Crabtree, I 989). It enhances glucocorticoid-induced apoptosis ; but inhibits activation-induced death of T-cell hybridomas - indicating that a block in the TCR-mediated signal transduction pathway can also trigger apoptosis (Zacharchuk et al., 1990). In isolated thymocytes, increase in the cAMP levels by independent mechanisms stimulated apoptosis. cAMP analogs that stimulated protein kinase A, also induced apoptosis. However, inhibitors of Ca2+and stimulators of protein kinase C both blocked apoptosis in response to cAMP suggesting interactions between the two protein kinase systems (McConkey et al., 1 9 9 0 ~ )Apoptosis . could be prevented by H7, an inhibitor of protein kinase C ; but not by H-A-1004, an inhibitor of CAMP-dependent protein kinase C (Ojeda et al., 1990). Lanotte et al. (1991) demonstrated that apoptosis can be induced solely by activation of CAMP-dependent protein kinase- I . It would appear from this review that the vast majority of cell types shown to respond to apoptotic stimuli are mostly of haemopoietic origin, known to be leading to terminal differentiation and death. Another class of cell types shown to be responsive to apoptosis are the cells from hormone-dependent tissues. Both the cell types possess several specialized receptors on the cell surface. Presence of specific active receptors is a prerequisite for signal transduction and perception of apoptotic stimuli. However, stimulation via specific receptor complex can rescue cells from hormone-induced death. On the other hand, cells that carry specific accessory molecules, can induce death in cells that recognise the accessory molecules through receptors. Crosslinking the accessory molecules enhance signal transduction and this in turn may lead to death. While T C R / C D 3 signalling induces death in a variety of haemopoietic cells, a block in TCR-mediated signal transduction can also include death in some cell types. It seems that an ordered sequential signal transduction within the basal levels is essential for viability of these cells. It is interesting to note that activation-induced and hormone-


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induced apoptotic pathways are mutually antagonistic and independent. It is the second messenger involved in the signal transduction process that clearly determines the fate of the cells. While stimulation of protein kinase C abrogates apoptosis, stimulation of protein kinase A induces programmation for death. The mechanism by which protein kinases determines the fate of the cells, and the biochemical steps involved in the process(es) remain to be elucidated in detail.

(d) Macromolecular synthesis Apoptotic cell death requires synthesis of RNA and proteins. In insects (Lockshin, 1969; Lockshin & Wong, 1981; Lockshin & Zakeri-Milovanovic, 1984) and in amphibians (Tata, 1966; Weber, 1969), where the organ undergoes apoptosis, as wall as in glucocorticoid-treated thymocytes (Munck, 1971; Voris & Young, 1981; Maytin & Young, 1983; Wyliie et al., 1984b), apoptosis could be prevented by inhibitors of synthesis of mRNA (actinomycin D) and protein (cycloheximide). Cell death in the epithelium of palate show features of apoptosis. Death is inhibited by epidermal growth factor but reinstated by treatment with dibutyryl CAMP (Pratt & Martin, 1975; Hassell & Pratt, 1977) and totally inhibited by blockade of protein synthesis (Pratt & Greene, I 976). Ecdysterone acts by activating specific genes within a differentiating cell (Fristrom et al., 1982; Riddiford, 1982) causing a transient synthesis of RNA and then an increase in incorporation of radioactive phosphate during the period RNA can be translated (Matsuura et al., 1968; Grzelak, Szczesna & Sehnal, 1982; Sehnal, Janda & Nemec, 1983; Lockshin & Shayani, 1983). Delay or abrogation of apoptosis by inhibition of macromolecule synthesis has been reported by a number of independent workers using systems like glucocorticoid-treated thymocytes (Cohen & Duke, 1984; Wyllie et ak., 1984b; McConkey et al., 1989b; MacDonald & Lees, I 990), cytotoxic T lymphocyte-mediated apoptosis (Cohen et al., 1985), y-irradiated lymphocytes (Sellins & Cohen, 1987; Yamada & Ohyama, 1988), activated T cells (Ucker, Ashwell & Nickas, 1989; Kizaki et al., 1989), activated T-cell hybridoma (Merc’ep, Noguchi & Ashwell, 1989; Shi et al., 1989) and rat myelocytic leukemia IPC-81 cell line (Lanotte et al., 1991). Similarly, inhibition of RNA and protein synthesis prevented death of nerve growth factor-deprived neuronal cells (Martin et al., 1988) and during regression of androgen-dependent tissues (Connor et al., 1988). The relationship between the change in ionic concentration and macromolecular synthesis is not yet defined. Glucocorticoids induced metallothionin synthesis in some mammalian cells (Karin & Herschman, 1979). Treatment of thymocytes with the hormone did not induce production of metallothionins (Maytin & Young, 1983). The role played by this class of proteins in apoptosis is still undefined. Transglutaminase has been associated with crosslinking of the cytokeratin components of the cytoskeleton. Apoptotic cells show an accumulation of e(y-glutamy1)lysine isopeptide in culture fluid and blood (Fesus et al., 1991). A number of enzymes are produced in cells undergoing apoptosis. Increase in steady state levels of RNA encoding for several enzymes such as transglutaminase (Piacentini et al., 1991a , b ) , ribonuclease (Engel, Lee & Grayhack, 1980), cathepsin D (Tanabe, Lee & Grayhack, 1982) and y-glutamyl transpeptidase (Schulte-Hermann et al., I 990) has been reported. Cyclic adenosine monophosphate kinase I (Lanotte et al., I 991) and degenerative enzyme, plasminogen activator (Rennie

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et al., 1984, 1988a) become active. While the precise biochemical role played by the enzymes in apoptosis has not yet been fully elucidated, the enzymes appear to be essential components of the cascade. Contrary to the concept the apoptosis may be delayed or inhibited by inhibition of macromolecular synthesis, there are only a few recent reports. Gliotoxin-induced apoptosis in macrophages does not require protein synthesis (Waring, I 990). Higher inositol triphosphate was probably related to the mobilisation of Ca2+for activation of endonuclease. Killer cells induce apoptosis without macromolecular synthesis (Clark et al., 1971 ; Thorn & Henney, 1976; Martz, 1977; Duke et al., 1983). Martin et al. ( I 990) have shown that in human promyelocytic leukemia HL-60 cells, presence of either cycloheximide or actinomycin D neither prevented nor delayed apoptosis. Furthermore, presence of either of the two chemicals induced large scale apoptosis in these cells. Mouse intestinal crypt cells (Searle et al., 1975; Ijiri & Potten, 1983, 1987), osteogenic sarcoma cells (Schwartz et al., I 973), avian leukemic myeloblasts (Heine, Langlois & Beard, I 966) and patient-derived B-chronic lymphocytic leukemia cells (Collins et al., 1991) show apoptosis when treated with actinomycin D or cycloheximide. Experiments with six other human cell lines confirmed that the phenomenon was not typical of these cells only (Martin et al., 1990). T h e results suggest that at least certain dividing cell populations do not require RNA or protein synthesis to undergo apoptosis. It is postulated that in these cells, effector molecule such as the endonuclease must be already present in the cell. Phosphorylation of the effector protein(s) and participation of the protein kinases might also be a key step controlling endonuclease activation. In these cells, some regulatory protein(s) may be needed to maintain control over the apoptotic cascade. ( e ) Gene activation At the molecular level, identification of the deathless mutants of Caenorhabditis elegans initiated the search for the mammalian equivalents of the cell death (ced) gene. In this nematode, a number of mutations affecting the process of cell death have been identified. These mutations have defined the genes ced-I, ced-a and nuc-I (Sulston, 1976; Hedgecock et al., 1983) that affect apoptosis. In particular, ced-3 and ced-4 genes may be involved in determining which particular cells express the fate of apoptosis during C. elegans development (Ellis & Horvitz, 1986). Studies with lymphocytes irradiated with y-rays provided further concept of activation of specific gene(s) during apoptosis. Use of cycloheximide or actinomycin D at various times after irradiation, delayed the apoptotic process. But once the metabolic process has initiated within the target cell, it proceeded to completion. This indicated triggering or activation of specific gene(s). Experiments with 5,6-dichloro I -P-Dribofuranosylbenzimidazole, a reversible inhibitor of RNA synthesis, showed that an inducing signal that is not simply mRNA persists within the irradiated cell (Sellins & Cohen, 1987). During apoptotic death of rat chloroleukemia cells at the half the maximal population density, the longer interspread repetitive D N A element (LIRn) suddenly becomes transcriptionally activated. Population growth was then inhibited. Within 24 h, after reaching maximal density, the population undergoes apoptosis. This is caused by sudden incorporation, apparently by retroposition via an RNA intermediate, of' about


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300000 copies of the LIRn element into random locations in the genome. T h e preceding growth inhibition was associated with repression to an undetectable level of c-ki-ras expression (Servomaa & Rytomaa, I 988). Gene activation has been reported during developmentally programmed death of Manduca sexta cells. cDNA clones for four genes have been isolated (Schwartz et al., 1990). Specific expression of cell death-associated gene products has been reported. Montpetit, Lawless & Tenniswood ( I 986) originally identified androgen repressed messages in involuting rat ventral prostate. A group of testosterone repressed mRNA sequences have been described (LCger, Montpetit & Tenniswood, 1987). Most abundant of these sequences is the testosterone repressed prostatic message-z (TRPMz), the translation product of which is a 46 kDa protein. TRPM-z gene product has been identified in a variety of cell types undergoing apoptosis (Buttyan et al., 1989). Transient but sharp rise of T R P M - z mRNA has also been reported during early phases of regression of hormone-dependent Schionogi mouse mammary carcinoma (Rennie et al., 1988b), rat prostate (Kyprianou & Isaacs, 1988; English, Kyprianou & Isaacs, I 989), in androgen-independent AT3 prostatic cancer cells undergoing apoptosis following treatment with 5-fluorodeoxyuridine and trifluorothymidine (Kyprianou & Isaacs, I 989), during regression of PC82 human prostate cancer following androgen ablation (Kyprianou, English & Isaacs, 1990), during induction of apoptosis of Lgzg tumour cells of C3H mice upon treatment with recombinant human tumour necrosis factor and inhibitors of DNA topoisomerase type I1 (Kyprianou, Alexander & Isaacs, 1991a) and also during death of estrogen-dependent MCF-7 human breast cancer cells xenografted on nude mice (Kyprianou et al., I 99 I b). Originally characterized in mammalian Sertoli cells as a constitutively expressed gene product, sulphated glycoprotein-z (SGP-z), has gained attention due to its rapid and sizeable induction in numerous types of mammalian cells undergoing apoptosis. SGP-z shares extensive sequence homology with TRPM-z and it has been shown only very recently that both the proteins are encoded by a single gene. Southern blot analysis of BglII restricted genomic DNA screened for the presence of restriction fragments homologous to SGPz indicated that the human homologue of SGP-z resides on chromosome 8 (Slawin et al., 1990). Using the regressing rat ventral prostate gland as a model to study androgen programmed cell death, a series of molecular events that accompany the process was characterized (Connor et al., 1988). Also, DNA and protein components of the nuclear acceptor sites for androgen receptors in rat prostate have been reported (Rennie et al., 1987). There is a sequential induction of specific gene transcripts. T h e first event in the cascade is an induction of specific gene encoding c-fos, the expression of which has been linked to perturbed intracellular Ca2+levels (Connor et al., 1988). This is followed by sequential induction of c-myc and 70 kDa heat shock protein mRNA (Wyllie et al., 1987; Barnes, 1988; Buttyan et al., 1988). T o test if Ca2+flux is an early physiological step involved in the apoptotic cascade, rats were simultaneously treated on castration either with verapamil or with nifedipine. Significant delay in regression was noted in both the experiments. Furthermore, the antagonists suppressed induction of transcripts encoding for both c-fos and TRPM-z (Connor et al., 1988); expression of c-H-ras and pSz also decreased (Kyprianou et al., 1991b). Recently it has been shown that an elevation of Ca2+, 3- 6-fold above the baseline, induced apoptosis in androgen-

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independent prostatic cancer cells (Martikainen et al., 1991). I t is probable that a sustained increase in Ca2' can play a major controlling role in activating specific genes needed for apoptosis. Recently wild-type p53 protein, a product of tumour suppressor gene, has been reported to induce apoptosis in murine myeloid leukemic M I line clone S6 (YonishRouach et al., 1991). Isolation and characterization of a cDNA clone from CEM C7 cells showed homology with the human HL- 14 gene encoding a /3-galactoside-binding protein. T h i s protein is over-expressed during glucocorticoid-induced programmed cell death. T h e mouse homologue acts as a potent cell growth inhibitory factor (Goldstone & Lavin, 1991). Very recently Cheng et al. ( I 992) demonstrated that tumorigenicity of T leukemia Be- I 3 cells, which lack endogenous p53 protein, can be suppressed when infected with a recombinant retrovirus encoding the wild type allele of human p53. Expression of p53 reduced growth rate, clonogenic potential and tumorigenicity of the cells. This opens the possibility of suppression/modulation of tumorigenic phenotype, at least in leukemic cells, through high-efficiency infection with retroviruses. T h e bcl-2 oncogene expression enhances the survival of B-cell precursors on withdrawal of growth factors. Myeloid precursors normally die by apoptosis (Williams et al., 1990). T h e finding that bcl-2 provides a survival advantage for the cells (Tsujimoto et al., 1985; Williams et al., 1990; Hockenbery et al., 1990; Cotter, 1990) by preventing the onset of apoptosis (Vaux, Cory & Adams, 1988; Tsujimoto, 1989) suggests that in some cells, programmed death may be negatively modulated by other genes. Involvement of the proto-oncogene bcl-2 in the prevention of apoptosis has been reported in T cells (Strasser, Harris & Cory, 1991),thymocytes (Sentman et al., 1991), germinal centre B cells and Burkitt lymphoma cells (Liu et a l . , 1991a , b ) and also in two pre-B-leukaemia lines (Alnemri et a l . , 1992). Recent evidences suggest that high level of bcl-2 enhances cell survival under conditions of repressed c-myc expression probably by mobilizing Ca2+ from the mitochondria to the cytoplasm. This in turn can cause activation of protein kinase C and help in enhanced survival (Alnemri et al., 1992). Epstein-Barr virus in circulating B cells may influence suppression of apoptosis by the expression of Epstein-Barr virus latent genes (Henderson et al., 1991) DNA transfection into human B cells demonstrated that expression of the viral membrane protein I suppresses apoptosis by up-regulating expression of the oncogene bcl-2. T h e mouse anti-Fas monoclonal antibody binds to a 36 kDa transmembrane receptor with significant homology with human tumour necrosis factor receptor, human nerve growth factor receptor and human B cell antigen CD40. T h e antibody, when recognized by appropriate cell surface antigen, can induce apoptosis either by preventing the activity of a factor necessary for survival or by serving as a positive death-inducing signal (Itoh et al., 1991). Methylation may be involved in activation/deactivation of a gene. Methylation is known to affect of DNA-protein interactions, regulation of gene expression, restriction endonuclease activity, replication and cell differentiation (Woodcock, Adams & Copper, 1982 ; Doerfler, 1983). Methylation is inhibited by the anticancer agent, 5-azacytidine. One subline (SAK 8) of murine thymocytes that is reported to possess functional glucocorticoid receptors but resistant to hormone-induced death (Gasson & Bourgeois, I 983), becomes sensitive to the hormone treatment when treated with 5-azacytidine.


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The DNA remains in a demethylated state following the drug treatment and the resistance of the cell correlates well with the level of methylation of DNA (Gasson, Ryden & Bourgeois, 1983) indicating thereby that certain genes must remain in a transcriptionally activated demethylated state for glucocorticoids to be effective. IV. INFLUENCE OF CELL CYCLE PHASE AND POSSIBLE MOLECULAR REGULATION

Little is known about molecular regulation and the factors influencing the onset of apoptosis. Chinese hamster V79 fibroblast cells when subjected to cold shock, undergo apoptotic death. Cells at the transition from exponential to stationary growth are most sensitive (Soloff et al., 1987). It was also shown that epidermal cells in their synthetic phase are more prone to uv-induced damage (Danno & Horio, 1982). One fundamental question that remains to be answered is whether apoptotic death can be induced at a specific point of the cell cycle progression. Unfortunately only scant information is available on this topic. While it is known that transcriptionally active chromatin is preferentially cleaved during apoptosis (Arends et al., 1990), Kung et al. (1990) reported absence of correlation between cell cycle phase at the time of anticancer drug addition and subsequent morphology of cell death. Treatment of L I Z I O / O cells with cisplatin (Sorenson et al., 1990) and murine BWg14 thymoma cells with dexamethasone or irradiation with y-rays (Kruman et al., 1991) showed inhibition of DNA synthesis correlated with arrest of the cells in the G, phase of the cell cycle. Inhibition of total RNA synthesis was observed initially. This was followed by a recovery that corresponded well with the passage of the cells through G, phase (Sorenson et al., 1990). DNA double-strand breaks appeared in cells destined to die (Sorenson & Eastman, 1988). Increase in poly(ADP-ribosy1)ation is commonly associated with DNA breaks (Murray & Kirschner, 1989a) causing a decrease in NAD pool and ATP levels. However, Sorenson et al. (1990) clearly demonstrated that poly(ADP-ribosy1)ation occurs after DNA degradation as an effect of the damage. Their studies showed that initial suppression of DNA synthesis during S phase followed by a recovery-reflecting a passage of the cells into G, phase. Cells were subsequently blocked at G, phase. This arrest could not be attributed to detectable changes in transcription. A probable explanation may be given from the RAD 9 gene function (Weinert & Hartwell, 1988; Hartwell & Weinert, 1989). The product of the gene is essential for arrest of cell division following DNA damage in Saccharomyces cerevisiae. As postulated previously by Tobey (1975), RAD 9 or its equivalent gene product may be involved in the surveillance mechanism. Sorenson et al. (1990) have shown that a cell may be lethally damaged yet continue to progress in the cell cycle and eventually destroy itself by apoptosis at G,M phase transition. Poisons of DNA topoisomerase type I1 may alter the activity of specific proteins (Gaulden, 1987; Lock & Ross, 199oa) and cause a block of cells at G, phase. Clear dissociation between drug-induced DNA topoisomerase I1 complexes and cytotoxicity has been reported (Estey et al., 1987; Chow & Ross, 1987; Chatterjee et al., 1990; Erba et al., 1992). Furthermore, toxicity of mAMSA and etoposide was reduced by concomittant treatment with cycloheximide ; but the drug-DNA-topoisomerase I1 complexes were not decreased (Chow & Ross, 1987 ; Schneider, Lawson & Ralph, I 989). Fragmentation of internucleosomal DNA, so typical of apoptosis, has been reported after administration of etoposide (Kaufmann,

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1989), teniposide and amsacrine (Walker et al., 1991). Here D N A cleavage had two distinct patterns : the first cleavage was due to drug-topoisomerase II interactions ; whereas the second cleavage was due to entry of the drug-treated cells into a phase of programmed death. It is probable that biochemical events that routinely occur at the G,/M phase transition may be involved in apoptosis. Studies during the past few years provided a better understanding of the events necessary for the passage of cells into mitosis (see, Hartwell & Weinert, 1989; Murray & Kirschner, 19896; O'Farrell et al., 1989; Pardee, 1989). A major regulatory protein is ~ 3 4 ~ ~ ~ ' k i nnecessary ase, for transition of cells from G, phase to mitosis, the activity of which remains inhibited during the onset of apoptotic cascade following . & Ross, administration of etoposide or y-irradiation (Lock & Ross, 1 9 9 0 ~ )Lock ( I 990 6 ) further demonstrated that the G, arrest following etoposide treatment is transient. Abnormally elevated ~ 3 4 ~kinase ~ " activity was detectable during mitosis, which was related to cell death. They postulated that a prolonged mitosis during which activities of specific cell cycle regulated proteins/regulatory proteins are altered, may result in D N A damage and cell death. T h e cdcz gene is activated and inactivated by specific phosphorylations and by a variety of associated proteins (Draetta & Beach, 1988; Pines & Hunter, 1989; Riabowol et al., 1989). Regulation of the gene is defined in yeast: while cdc2j activates the kinase (Strausfeld et al., 1991), weel suppresses it. Cells can undergo a catastrophe when mutation causes imbalance in these genes (Russell & Nurse, 1986). D N A damage alone may not be a prerequisite for cell death. I n human cells, the ~ 3 4 kinase ~ ~ ~complex ' interacts directly with p13 (Draetta & Beach, 1988). Microinjection of p I 3 or antibodies to p I 3 to rat fibroblasts causes micronucleation and death (Riabowol et al., 1989). Kung and co-workers (1990) suggested that cytotoxicity does not derive directly from the specific biochemical action of the drug per se: but results from the disparate inhibition of certain cell cycle processes or from dissociation of normally integrated and linked cell cycle events. In support of their hypothesis, the authors provided examples of apoptotic death of Chinese hamster ovary cells (strain AA8) treated with aphidicolin or vincristine. Further support of the hypothesis comes from the studies of Lau & Pardee (1982) and Schlegal & Pardee (1986, 1987) who reported potentiation of cytotoxicity of cells treated with caffeine to uncouple cell cycle events leading to mitosis or by uncoupling protein synthesis from D N A synthesis (Zetterberg, Engstrom & DafgHrd, 1984; Larson et al., 1986). Enoch & Nurse (1990) provided additional support. Their studies demonstrated that in the fission yeast, Schizosaccharomyces pornbe, cdcag mutants which are defective in the control of mitosis, are highly susceptible to the lethal effect of D N A synthesis inhibition. Presence of an additional mutation at cdclo gene (preventing cells from entering start) killed only those cells that entered S phase prior to DNA synthesis inhibition. T h e result supports the concept that an ordered progression of cell cycle events is an essential prerequisite for cellular viability. Indeed, little is known about the molecular regulation involved during the induction of apoptotic death. Unfortunately no definite regulatory mechanism can be held responsible. A full understanding of the process would be essential to design compounds for successful killing of cancer cells. New informations are warranted to induce death in tumour cell population under physiologic conditions.


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V. SIGNIFICANCE OF APOPTOSIS

Apoptosis is a cell elimination mechanism involved in a variety of physiological conditions such as embryogenesis (Wyllie, 1981; Allen, 1987), growth factor deprivation (Araki et al., 1990), hormonal regulation of homeostasis (Nawaz et al., 1987) and maintenance of homeostasis of the immune system (Duvall & Wyllie, 1986). It regulates turnover of cells in vivo (Saaraf & Bowen, 1986; Pierce, Lewellyn & Parchment, 1989), in vitro (Lynch, Nawaz & Gerschenson, 1986; Cotter & Martifi, 1989) and in organs (Busch, Winterhager & Fischer, 1986; Samia et al., 1987; Parr, Tung & Parr, 1987; Tessitore et al., 1989). It operates during ageing (Newman, Henson & Henson, 1982; Savill, Henson & Haslett, 1989; Savill et al., 1989; Martin, Bradley & Cotter, 1990); is a major component of normal erythropoiesis (Koury & Bondurant, 1990) and human retina development (Penfold & Provis, 1986). A wide variety of cell specific stimuli, usually of low intensity, can trigger on the mode of self destruction. Studies have confirmed that the cells are capable of initiating a programme for death under physiological condition without affecting the neighbouring cells. The most outstanding feature of apoptosis is destruction of the entire genome by the action of endonuclease. This guarantees termination of transcription and prevents accidental contribution of a partially damaged genome or a genome infected with virus (Terai et a l . , 1991) to the daughter cells or to the neighbouring cells while undergoing phagocytosis. Apoptotic cascade of events results in chromatin cleavage and condensation of nuclear bulk permitting the cells to dissociate into several small packages (apoptotic bodies) of a size suitable for phagocytosis and easy removal. The relatively recent observation that apoptosis has a potential to check cancer growth (Szende, Zaltnai & Schally, 1989; Trauth et al.,1989; Yuh & Thompson, 1989; Lanotte et al., 1991) and that a variety of antineoplastic agents of varied nature and of very different mechanism of action can induce apoptosis is of importance. A proper understanding of the biochemical events associated with the apoptotic cascade and knowledge of molecular regulation of the death process may help in drawing successful new strategies to focus the action of chemotherapeutic agents specifically against cancer cells in the body. VI. SUMMARY AND CONCLUSIONS

Programmed cell death or apoptosis occurs under physiological conditions as a result of physiological effectors. It is a relatively slower process and requires active participation of the cell in the suicidal mechanism. Apoptosis is controlled by precise intrinsic genetic programme and may be induced by almost all those stimuli causing necrosis. The role played by the intensity in determining the death process and the underlying mechanism is imperfectly understood. Morphologically apoptotic cells appear as small condensed body. The chromatin is dense and fragmented, packed into compact membrane-bound bodies together with randomly distributed cell organelles. The plasma membrane loses its characteristic architecture and shows extensive blebbing. It buds off projections so that the whole cell may split into several membrane-bound apoptotic bodies. Significant chemical changes take place in the plasma membrane. This helps in recognition of the apoptotic bodies by phagocytes. At this moment it is unclear if all cells can undergo apoptosis or it is a characteristic of only some tissues which are predisposed to apoptotic death being

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directly under the control of hormones or growth factors. Experimental studies aimed at comparison of induction of apoptosis in cells of different origin are warranted to elucidate this point. Biochemically a pre-commitment step for induction of death programmation through macromolecular synthesis is essential for most systems. T h e double-stranded linker D N A between nucleosomes is cleaved at regular inter-nucleosomal sites through the action of a Ca2+,Mg2+-sensitiveneutral endonuclease. Zinc is a potent inhibitor of the enzyme. Calcium probably plays a key controlling role in activation of the enzyme since prevention of Ca2+ increase prevents endonuclease activation. It is becoming evident that signal transduction through appropriate receptors control the Ca2+flux in the cells. Most apoptotic cells require synthesis of RNA and proteins. Delay or abrogation of apoptosis by inhibition of macromolecular synthesis is well known. T h e dying cells show high mRNA levels for several enzymes. Several degradative enzymes become active. Regulatory proteins maintain control over the apoptotic cascade. At the molecular level, search has been initiated for the mammalian equivalents of the cell death (ced) gene. Activation of several specific genes is indicated. Specific expression of cell death-associated gene products (e.g. TRPM-z/SGP-z) has been reported in several unrelated apoptotic cell systems. Sequential induction of c-fos, cmyc and 70 kDa heat shock protein is reported. Studies demonstrate that certain genes must remain in a transcriptionally active demethylated state during programmed cell death. Recent evidences clearly indicate that apoptosis may be positively or negatively modulated by certain genes. Only scant information is available about the influence of cell cycle phase on apoptosis. Recently it has been shown that cells are initially arrested at G, phase of the cell cycle. Programmed cell death occurs when the cells pass through the G,M phase transition. Abnormally elevated p34cde2kinaseactivity was detected at this point. It is possible that biochemical events that routinely occur at this stage may be involved in control of apoptosis. Several gene products may be involved in the surveillance mechanism. T h e importance of apoptosis in organ development during embryogenesis does not need to be stressed. It is the major mechanism of regulation of homeostasis of a number of physiological systems in the body that ensures easy elimination of the cells by phagocytosis under physiological conditions. T h e recent observation that a variety of antineoplastic agents of very different mode of action can induce apoptosis, provides a new concept in the field of anticancer drug development. A full understanding of the biochemical events and molecular regulation may help in drawing new strategies to focus the action of anticancer agents especially against cancer cells in the body. Unfortunately difficulties exist with the identification and quantitative measurement of apoptosis, particularly during the early phases of onset of death. Cell viability detection by vital dye exclusion or measurement of cell volume shrinkage during the early phases proved to be unsatisfactory. T h e typical chromatin cleavage pattern, as seen by agarose gel electrophoresis, remains to be the only reliable method. At a later stage, depending on the severity of the stimulus, apoptotic death is often mixed with necrotic death. Electrophoretic studies then show a streak and the typical regular cleavage of D N A in multiples of 180-200 base pairs (the ladder pattern) is impossible to achieve since these fragments are mixed with fragments of variable lengths from


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necrotic cells. Furthermore, electrophoretic studies provide a qualitative, rather than a quantitative measure. Quantitative estimation in terms of presence of small dense apoptotic bodies in a sample, particularly in tissues, is difficult owing to their rapid removal by phagocytosis. It is the inability to detect and quantitate apoptosis at an early stage which provide a formidable block of rapid elucidation of biochemical and molecular mechanisms of programmed cell death. More efficient methodologies should be developed to counteract this problem. VII. ACKNOWLEDGEMENTS The author is grateful to Professor A. K. Sharma, D.Sc., FNA, Indian National Science Academy Golden Jubilee Professor & Programme Coordinator, University of Calcutta, and to Dr. M. D’Incalci, MD, Chief, Laboratory of Cancer Chemotherapy, Istituto di Ricerche Farmacologiche ‘Mario Negri ’, Milan for critical comments and counsel with the manuscript; to Ms. S. D. De, Queen Mary College, University of London, for her help in manuscript preparation ; to Drs. Kalpana Agarwal (University of Calcutta), Madhumita Joardar Mukhopadhyay (Universit6 Laval) and Mauro Ponti (ICI Italia, SPA, Milan) for kindly providing photocopies of several papers. The work was partially supported by Pool Scheme, Government of India.

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