Indian Journal of Biotechnology Vol I, January 2002, pp. 58-72

Apoptosis: An Overview

Rana Anjum and Ashok Khar*

Centre for Cellular & Molecul ar Biology, Uppal Road, Hyderabad 500007. India

Apoptosis, a highly ordered cascade of enzymatic events that culminates in cell death and entails the autolytic degradation of cellular components, is characterized by blebbing of cell membrane, nuclear and cytoplasmic condensation, protein fragmentation and DNA degradation followed by rapid engulfment of cell corpses by neighbouring cells. It is essential for maintenance of cellular homeostasis and deregulation of this process leads to a spectrum of pathological manifestations. Caspases, which form the proteolytic network within the cell are among the critical components of cell death process. They bring about the cleavage and degradation of a number of proteins that result in cell demise. Mitochondria are regarded as the central control point in the execution of apoptosis. They release a potent cocktail of pro-apoptotic proteins into the cytosol, which results in the amplification of the death cascade, the most prominent one being cytochrome c. The mechanism by which cytochrome c is released into the cytosol is controversial but seems to be regulated by the Bcl~2 family of proteins.

Keywords: apoptosis, programmed cell death mechanisms, caspases, mitochondria

Introduction Apoptosis or programmed cell death is a geneti­

cally programmed, highly conserved, intricate mechanism of cellular suicide, that can be triggered by a variety of physiological and pathophysiological conditions and which plays a crucial role in the development and maintenance of cellular homeostasis in multicellular organisms (Arends & Wyllie, 1991; Wyllie et ai, 1980). It is essential for normal development and de-regulation of the process leads to a spectrum of defects ranging from embryonic lethality , tissue specific perturbation of post-natal development, and a high susceptibility to cancer (Thompson, 1995). Apoptosis is invoked in a wide variety of different biological systems, including normal cell turnover, development of the immune system, embryonic development, metamorphosis, growth factor withdrawal, exposure to chemotoxins or even physical damage (Ellis et ai, 1991). Apoptotic cell death is characterized by a series of distinct morphological and biochemical alterations which include cell shrinkage, membrane blebbing, chromatin condensation and fragmentation, formation of apoptotic bodies that are rapidly devoured by phagocytes (Thompson, 1995).

"Author for correspondence: Fax: +91-40-7160591 , 716031 i Emai l: khar@ccmb.ap.nic.in; khar@gene.ccmbindia.org

Historical Perspective The phenomenon of cell death occurring as a

normal part of both development and homeostasis was established more than forty years ago (Gli.icksmann, 1950). In vertebrates, cell death has been observed in almost all tissues and has been studied more extensively in the developing nervous system (Hamburger & Oppenheim, 1982) and in the immune system (Duvall & Wyllie, 1986).

In the early 1970s, Kerr, Wyll ie and Curie found that cells undergo at least two distinct forms of death: the well characterized and usually necrotic tissue damage induced by trauma, and a more protracted and morphologically distinct form of cell death which they termed 'apoptosis' (Wyllie et ai, 1980). In archaic Greek, apoptosis refers to leaves falling off trees in the autumn. The word was chosen to suggest cell loss that is desirable for the survi val of the host (Touchette & Fogle, 1991). On the contrary, the term 'programmed cell death' (PCD) was used to describe cell death that occurred in predictable places and at predictable times during development, to emphasize that the death is somewhat programmed into the developmental plan of the organism. Using this strict definition, cell death during cyclic or seasonal involution of gonads , turnover of epithelia and hematopoietic cells, morphogenesis in embryonic development, metamorphosis , and a few other examples is clearly programmed. While the term

ANJUM AND KHAR: APOPTOSIS-THE CELL DEATH PROCESS 59

'apoptosis' etymologically evokes a programmed occurrence in the organism, the current tendency is to use it in reference to the morphological changes accompanying this type of cell death which includes cytoplasmic and nuclear condensation/fragmentation. There are examples of truly programmed cell death that is not apoptosis by morphology (Schwartz & Osborne, 1993). But often both these terms are used interchangeably.

The existence of an intrinsic cell suicide program was ascertained through genetic studies carried out by Horvitz and colleagues in the nematode, Caenor­habditis elegans, that identified genes involved in the cell death program and its control (Ellis & Horvitz, 1986), and found out that some of these genes were homologous to mammalian genes (Hengartner & Horvitz 1994). These studies demonstrated very early occurrence of PCD in the course of metazoan evolution and the substantial conservation of its basal machinery from nematodes to humans.

Characteristic Features of Apoptotic Cells Apoptosis is characterized by a distinctive and

orchestrated sequence of morphological changes . .In the initial phase, an individual cell embedded' in normal tissue loses contact with its' neighbours (Wyl lie et ai, 1980). The cell shrinks due to loss of cytoplasmic volume (shrinkage necrosis) and condensation of cytoplasmic proteins. The nuclear chromatin gets condensed resulting in the fragmentation of cellular DNA. The second phase is characterized by membrane ruffling and bleb bing, leading to cellular fragmentation and formation of apoptotic bodies, frequently contaInIng nuclear remnants (Williams et ai, 1974) (Fig. la). While most of these events are taking place, the cell still excludes vital dyes and retains most of its internal constituents. In the final phase, the neighbouring cells and macrophages phagocytose the fragments for complete degradation.

DNA Degradation in Apoptosis Induction of apoptosis is characterized by the

internucleosomal DNA cleavage, a biochemical event used as a definitive apoptotic marker (Schwartzman & Cidlowski, 1993). This pattern of DNA degradation occurs by activation of an endogenous endonuclease that cleaves the DNA in the linker region between h~stones on the chromosomes. Since the DNA wrapped around the histones comprises -180-200 bp, multiples of this fragment are characteristically

observed and are commonly referred to as the 'apoptotic ladder' (Fig. 2). Despite overwhelming evidence in favour of internucleosomal DNA cleavage activity, as a characteristic of apoptosis, several accounts of apoptosis in the absence of this DNA- cleavage pattern have been reported (Zakeri et ai, 1993; Boe et ai, 1991), Cleavage of DNA into large fragments of 50-300 kb during apoptosis have also been reported (Walker etal, 1991).

Fig. I-Phosphatidyl serine externalisation and annex in- V binding in cells undergoing apoptosis (b). Control cells (a) do not show annexin-V binding. Nuclear morphology is revealed by Hoechst staining. Cells undergoing apoptosis show the formation of nuclear bodies (b).

Fig . 2-Fonnation of an oligollllleosomal DNA ladder in cells undergoing apoptosis. a hallmark of apoptosis. Lane I .. control ce ll s. lane 2 .. cells treated with an apoptotic inducer.

60 INDIAN J BIOTECHNOL, JANUARY 2002

One of the main nucleases involved in apoptotic DNA degradation is caspase-activated DNase (CAD) (Enari et ai, 1998). CAD is normally complexed to its chaperone and inhibitor, ICAD. In dying cells, ICAD is cleaved by caspases, releasing the inhibition on CAD. However, CAD-deficient cells fail to undergo oligonucleosomal DNA fragmentation, if stimulated to undergo apoptosis (Liu et ai, 1998) but still exhibit other features of apoptotic cell death. The sequence of murine ICAD is highly homologous to a human protein, DFF45, which also contains caspase-3 sites and is cleaved during apoptosis. DNA degradation may serve to limit potentially dangerous genetic information spreading from the dying cells and also to avoid persistence of auto-antigenic material such as nucleosomes, which may be involved In the pathogenesis of auto-immune disorders.

Phagocytosis of Apoptotic Cells One of the striking features of apoptotic cell death

is the rapid engulfment of the dying cells by phagocytes and the complete elimination of cell corpses , thus protecting the sUlTounding tissue from the damaging effects of released intracellular contents. Phagocytosis is triggered by physiochemical changes in the plasma membrane of the dying cells that function as "eat me" signals, enabling the hetero­phagic recognition and engulfment of the apoptotic cells by adjacent healthy cells. One of the well­characterized signals for phagocyte recognition of apoptotic cells is the loss in plasma membrane phos­pholipid asymmetry (Fadok et ai, 1998). Viable cells maintain an asymmetric distribution of different phospholipids with phosphatidyl choline and sphingo­myelin being present on the outer leaflet, and phos­phatidylethanol amine and phosphatidylserine (PS), restricted to the inner leaflet of the membrane.

It has been demonstrated that cells undergoing apoptosis expose PS on the outer leaflet of the plasma membrane (Fadok et ai, 1992). This cell surface exposed PS, functions as a tag for specific recognition by macrophages and for phagocytosis of the dying cell, detected by annexin V, a specific PS binding protein (Fig. 1 b). This phenomenon has been shown to be an early event during apoptosis, initiated at a ti me follow ing the caspase proteolytic cascade but possibly preceding nuclear condensation and breakdown of intracellular cytoskeletal and nuclear matrix constituents (Martin et ai, 1995) .

Caspases: The Executioners of Apoptosis Caspases (Cysteinyl aspartate-specific proteinases)

mediate highly specific proteolytic cleavage events in dying cells which collectively manifest the apoptotic phenotype (Alnernri et ai, 1996). The key and central role that these enzymes play in a biochemical cell­suicide pathway has been conserved throughout the evolution of multicellular organisms. Caspases were implicated in apoptosis with the discovery that CED-3, the product of a gene required for cell death in the nematode, Caenorhabditis elegans, is related to mammalian interleukin 1 ~-converting enzyme (ICE or caspase-l) (Thornbeny et ai, 1992). More than a dozen caspases have been identified in humans, about two-thirds of these have been suggested to function in apoptosis (Thornbeny & Lazebnik, 1998). The cunent nomenclature as well as the trivial names of some of the caspases is shown in Table 1.

Structure, Activation and Regulation of Caspases Caspases are synthesized as inactive pro-enzymes

which are activated by cleavage adjacent to aspartate residues to liberate one large and one small subunit, which associate into an CX2~2 tetramer to form the active enzyme. The absolute requi rement for cleavage next to aspartate enables caspases to activate other caspases, thereby setting the stage for an amplifying cascade (Fig . 3). Recognition of at least four amino acids NHrterminal to the cleavage si te is also a necessary requirement for efficient catalysis. The preferred tetrapeptide recogl1l tlOn motif differs significantly among caspases and explains the diversity of their biological activities (Thornberry et ai, 1997). Current understanding of caspase function

Table l-Caspase nomenclature

Current name Triv ial name

Caspase- l ICE Caspase-2 Ich- IL' NEDD2 Caspase-3 CPP32,Apop~n,Yama

Caspase-4 TX lch-2, ICE rei-II Caspase-5 ICErclJl1. TY Caspase-6 Mch 2 Caspase-7 Mch 3, CMH- I , ICE-LAP3 Caspase-8 Mch5, MACH, FLICE Caspase-9 Mch6, ICE-LAP6 Caspase-IO Mch4, FLlCE-2 Caspase- Il mICH-3 , mCASP-I I Caspase- 12 mICH-4, mCASP-12 Caspase- 13 ERICE Caspase- 14 MICE

ANJUM AND KHAR: APOPTOSIS-THE CELL DEATH PROCESS 61

Death r.eceptors

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ProteoJysis of ubstrates

Fig. 3-Schematic representation of the general pathway of apoptosis

has been facilitated by the development of cell-free systems for the study of apoptosis (Lazebnik et ai, 1993; Anjum & Khar, 1997). Such cell-free systems rely on the addition of different components to isolated nuclei in vitro, fo llowed by the quantitation of nuclear changes in vitro. Agents triggering cytosolic and nuclear apoptotic changes in such an artificial system include cy tosols from cells under­going apoptosis (which contain activated caspases) or cytosols from normal cells supplemented with recombinant caspases. Cell-free model s share several advantages over the conventional methods for the

dissection of the apoptotic pathway , owing to the rapidity of the experiments and the ability to add and remove proteins and other reagents that may not normally traverse the cell membrane.

Cellular Targets of Caspases The role of proteases in cell suicide is to disab le

critical homeostatic and repair processes, as well as to cleave key structural components, resulting in the systematic and orderly disassembly of the dying cell. Several important caspase substrates have been identified in recent years. Polypeptides known to be

62 INDIAN J BIOTECHNOL, JANUARY 2002

cleaved during apoptosis include enzymes involved in genome function such as poly (ADP-ribose) polymerase (Lazebnik et ai, 1994), the catalytic subunit of DNA-dependent protein kinase (Emoto et ai, 1995), the 70-kDa protein component of the U 1 small ribonucleoprotein (Tewari et ai, 1995), ICAD (Sakahira et ai, 1998) etc. Cleavage of nuclear lamins is required for nuclear shrinkage and budding (Rao et aI, 1996). Loss of overall cell shape is probably caused by the cleavage of cytoskeletal proteins such as fodrin and gelsolin (Kothakota et ai, 1997), p21_

activated kinase-2 (PAK-2) (Rudel & Bokoch, 1997) and many more. Caspases thus help to cut off contacts with surrounding cells, reorganize the cytoskeleton, shut down DNA replication and repair, disrupt the nuclear structure and disintegrate the cells into apoptotic bodies .

Inhibitors of Caspases Several classes of natural as well as synthetic

caspase inhibitors have been described that can subvert the cell suicide program. These include:

(i) Bcl-2 family of proteins. Bcl-2 was first isolated as the proto-oncogene involved in t(l4; 18) translocation found in human follicular lymphomas (Bakshi et ai, 1985). Bcl-2 and related family members, such as Bcl-XL, inhibit cell death induced by many stimuli (Vaux et ai, 1988). Several recent studies suggest that Bcl-2 and Bcl-XL exert their anti­apoptotic action at or before the processing of certain caspases to their catalytically active forms (Chinnaiyan et ai, 1996).

(ii) CrmA, p35, cellular lAP's . These are the naturally OCCUlTing caspase inhibitors . Cytokine response modifier (CrmA) is a 38 kDa serpin from Cox pox virus that appears to facilitate viral infection through both inhibition of the host inflammatory response and inhibition of apoptosis (Ray et ai, 1992). CrmA has been evaluated as an inhibitor of both caspase-l and caspase-8 (Xue & Horvitz, 1995).

p35, a 35-kDa protein from Baculovirus also appears to attenuate apoptosis through inhibition of caspases (Deveraux et ai, 1997). Synthesis of p35 has been shown to prevent cell death in insect cells, in C. elegans and in mammalian systems.

The lAP (inhibitor of apoptosis) gene family, comprise a third group of polypeptides that prevent cell death in a variety of species. One family member, XIAP, has been shown to be a direct inhibitor of caspase-3 and -7 (Villa et ai, 1997).

(iii) Synthetic peptide inhibitors. Synthetic peptide inhibitors that mimic cleavage sites of the caspases (usually at an Asp-X bond) have been used for in vitro and in vivo analysis of enzyme activity (Garcia­Calvo et ai, 1998). These peptides are generally very small, 3 to 4 amino acids in length and include zVAD-fmk (broad spectrum inhibitor), YV AD-cmk (caspase-l inhibitor), DEVD-CHO (caspase-3 inhibi­tor), etc. The sequence of these various peptides is based on the caspase recognition sites of substrates. In zV AD-fmk, due to an aspartate residue mimicking the cleavage site and a fmk group, forming a covalent inhibitor I enzyme complex, the inhibitor instantly and irreversibly binds to the catalytic site of caspases (Kostura et ai, 1989). These peptide inhibitors are soluble, relatively stable and show dose-dependent inhibition of cell death after induction with several different stimuli. These inhibitors have been used to order the upstream and downstream events within a particular apoptotic signaling pathway.

Genetic Analysis of the Biological Role of Caspases The role of individual caspase in apoptotic process

has been deciphered by the targeted di sruption of these enzymes in mice. It was observed that no caspase knockout described till date abolished all apoptosis during development and for any caspase, defects in apoptosi s are both cell type and stimulus­dependent.

Caspase-l. ICE/caspase-l is the cysteine protease responsible for the proteolytic conversion of the 31 kDa inactive cytokine precursor, pro-interleukin I~ ,

to its 17.5 kDa active form, a key mediator of inflammation (Miura et ai, 1993). Caspase-l was shown to share 28% sequence identity to C. elegans CED-3 and shown to play a role in apoptosis (Thornberry et ai, 1992). Though overexpression of ICE in many experimental systems results in increased apoptosis (Kuida et ai, 1995), the results of ICE knockout experiments rule out the possibility of the involvement of ICE as an indispensable candidate in apoptosis (Kumar et ai, 1994). Caspase-l·l . mice are developmentally normal , however caspase- r /-cells have been reported to be more resistant to apoptosis induced by CD95 ligation (Kumar et ai, 1994).

Caspase-2. Nedd2 was originally identified as a developmentally downregulated gene in mouse brain (Bergeron et ai, 1998). Caspase-2 deficient mice are developmentally normal, and their cells undergo a

ANJUM AND KHAR: APOPTOSrS-THE CELL DEATH PROCESS 63

normal apoptotic process in response to various stimuli, including NGF (Wang et ai, 1994). The human homologue named Ich-l was isolated from a human fetal brain cDNA library and shown to encode a protein with sequence homology to ICE and ced-3 (Khar et ai, 1997). Overexpression of caspase-2 has been shown to induce apoptosis in tumor cells (Kuida et ai, 1996). The inhibitor profile and the substrate specificity of the Nedd2/ lch-l gene product have not been explored systematically.

Caspase-3. Caspase-3 is one of the key executioners of apoptosis being responsible either partially or totally for the proteolytic cleavage of many key proteins, such as the nuclear repair enzyme PARP (Lazebnik et at, 1994). Caspase-3 is present in cells as a 32 kDa pro-enzyme that is cleaved to 17 kDa active form upon apoptosis (Xue & Horvitz, 1995). Caspase-3 prefers a DXXD-like substrate whereas caspase-l prefers a YV AD-l ike substrate (Xue & Horvitz, 1995). Caspase-3 knockout mice can survive to birth but they exhibit perinatal mortality as a result of defects in brain development that correlate with reduced apoptosis (Boldin et at, 1996).

Caspase-S. Caspase-8 is an upstream caspase involved in the triggering of death cascade in Fas and TNF-a mediated apoptosis. It has an N-terminal domain with marked identity to the death effector domain (DED) of the adaptor molecule, FADD/ MORTI (Fas-associating protein with death domain) which interacts with CD9S and TNF receptor (Muzio et at 1996; Varfolomeev et ai, 1998). Over-expression of caspase-8 results in apoptosis, and mutation of its catalytic cysteine residue abolished its apoptotic potential Caspase-8 knockout mice develop normally until embryonic day 11.5 and then begin to die. Histological examination revealed poorly developed heart musculature (Kuida et at, 1998).

Caspase-9 (/CE-LAP6/mch6) . Caspase-9 is a member of the CED-3 subfamily , bearing high simi larity to caspase-3. The major difference between caspase-9 and other family members is the active site pentapeptide QACGG, in which a Glycine is found instead of the usual Arginine. Overexpression of caspase-9, but not of a mutant in which the catalytic Cysteine was replaced with an Alanine, induced apoptosis in MCF7 cells. Caspase-9 knockout mice show abnormalities in brain development similar to those of caspase-3 knockout animals (Hakem et ai, 1998).

Caspase-Independent Cell Death Caspases are required for the complete manifes­

tations of apoptotic morphology, yet are dispensable for cell death to occur in many systems. Over­expression of Bax or Bak induces apoptosis in the presence of broad-spectrum caspase inhibitors (McCarthy et at 1997). They also induce mitochon­drial dysfunction and kill yeast, which lack endogenous caspases (Muzio et at, 1996). Even fas­mediated cell death, a paradigm of receptor-mediated primary caspase activation (Vercammen et at, 1998), is only retarded but not prevented by caspase inhibition, at least in L929 cells (Susin et at, 1996). AIF undergoes mitochondrio-nuclear translocation which is caspase independent, resulting in apoptosis (Jacobson et ai, 1993). These observations underline the probable role of caspase-independent death mechanism. Caspases are required for the complete manifestation of apoptotic morphology, yet are dispensible for cell death to occur in many systems.

Mitochondria-the Central Regulatory Point in Apoptosis

Mitochondria were regarded as pass ive organelles that support life, but play no role in apoptosis . The absence of changes in mitochondrial ultrastructure as well as the fact that cells lacking mitochondrial DNA can undergo apoptosis (Vander Heiden & Thompson , 1999) initially supported the idea that mitochondria do not participate in the process of apoptosis. Recently there had been a paradigm shift in the area of apoptotic research, and the key role, this organelle plays during cell death is now getting unraveled.

The first evidence emphasizing the role of mitochondria in apoptosis ca.me from the studies by Newmeyer et at, who demonstrated using a cell-free system, the requirement of mitochondria-enriched heavy membrane fractions , in Xenopus egg extracts, for the apoptos is to occur. The fact that . members of the Bcl-2 family of proteins regulate cell death, by altering mitochondrial homeostasis (Mayer et at, 1995) also suggested a key role for mitochondria in apoptosis (Fig. 4).

Release of Death Promoting Factors from Mitochondria during Apoptosis

Cell death is characterized by the release of various proteins that are normally sequestered in the intermembrane space of mitochondria into the cytosol. Depending on the stimulation, this event can

64 INDIAN 1 BIOTECHNOL, JANUARY 2002

Jj~I>" daM'IIe

'~ ".p~J

'~

~

Fig. 4-The mitochondrial-dependent and - independent pathway in mammalian eels

lead to necrosis through irreversible mitochondrial damage and energetic catastrophe or to apoptosis through caspase activation.

(i) Reiease of Cytochrome c. Cytochrome c is encoded in the nucleus and translated on cytoplasmic ribosomes as apo-cytochrome c and follows a unique pathway into mitochondria that does not require the signal sequence, electrochemical potential and general protein translocation machinery (Kluck et ai, 1997) . The apoprotein in the intermembrane space combines with heme to become the mature protein. Cytochrome c functions as an electron carrier in oxidative phosphorylation, shuffling electrons from complex III to complex IV.

Wang and colleagues demonstrated that addition of dATP to the cell-free extracts from HeLa cells resulted in typical apoptotic changes including activation of caspase-3 and nuclear DNA fragmen­tation (Liu et ai, 1996). Fractionation of the extracts revealed the existence of two factors necessary for the above effect. One of the required factors surprisingly proved to be cytochrome c (Liu et ai, 1996). Subse-

guent investigations demonstrated that following ~xposure of cells to apoptotic stimuli , cytochrome c is rapidly released from mitochondria to cytosol (Yang et ai, 1997).

The other factor fractionated from the extracts was the protein Apaf-l (apoptosis-activating factor 1), a CED-4 homolog in C. elegans (Li et ai, 1997). Apaf-l nas an amino terminal caspase-recruitment domain (CARD) and also possesses nucleotide binding site. It was shown that addition of Apaf-l, cytochrome c (Apaf-2) and dATP to pro-caspase-9 (Apaf-3) results i.n conversion of pro-caspase-9 to active caspase-9. Caspase-9 causes cleavage of pro-caspase-3 to ~aspase-3, the key enzyme involved in apoptosis (Zamzami et ai, 1996).

(ii) Release of apoptosis inducing factor (A IF). Mitochondria isolated from apoptotic cells were demonstrated to induce apoptosis in the acellular system. These results emphasized that mitochondria can effectively control nuclear apoptosis (Susin et ai , 1999). The mitochondria that undergo permeability transition (PT) would liberate a protein (AIF) capable of inducing nuclear apoptosis (Jacobson et ai, 1993). AIF is normally confined to mitochondria, yet subject to mitochondrio-nuclear translocation upon induction of apoptosis by diverse agents. This nuclear localization of AIF is compatible with the presence of several putative and nuclear localization signals within AIF. The mitochondrio-nuclear translocation of AIF is caspase-independent. When added to purified nuclei from HeLa cells, recombinant AIF protein induces DNA loss, peripheral chromatin condensation and digestion of chromatin into -50 kbp fragments but no oligonucleosomal fragmentation is observed (Du et ai, 2000).

(iii) Reiease of SMACIDIABLO. Recent investigations identified a mammalian lAP inhibitor, known as Smac (second mitochondria-derived activator of caspases) (Verhagen et ai, 2000) or DIABLO (direct lAP-binding protein with low pI) (Loeffler & Kroemer, 2000) . Smac/DIABLO is a mitochondrial protein, but is released into the cytosol in cells induced to die, presumably following the same exit route as cytochrome c. It binds to lAP family members and neutralizes their anti-apoptotic activity.

Mechanism of Cytochrome c Release The exact mechanism resulting in the release of

cytochrome c to cytosol is still controversial, but it is

ANJUM AND KHAR: APOPTOSIS-THE CELL DEATH PROCESS 6S

evident that Bcl-2 family is intimately involved in the regulation of this process. Two competing models have been proposed to explain how cytochrome c is released fro m mitochondria during apoptosis.

(i) The first model postulates the opening of a megachannel called the permeabi lity transition pore (PTP); which is a large conductance, cyclosporin­inhibited channel. This channel is poorly characterized but is proposed to span both the inner and outer mitochondrial membranes at sites at which the two membranes are apposed. The adenine nucleotide translocator (ANT), located in the inner membrane, the voltage-dependent anion channel (VDAC) found in the outer membrane and the matrix localized cyclophilin D are considered to be major components of the PTP. Bax was also found to be a part of this complex (Marzo et aI, 1998). According to this model , PTP openers, including Bax cause permeabilization of the inner membrane and mitochondrial depolarization by binding to the ANT (Petit et aI, 1996). This process allows entry of water and solutes into the matrix, leading to the swelling and rupture of the outer mitochondrial membrane allowing cytochrome c and other proteins to be released passively (permeability transition) (Eskes et aI , 1998).

However, several observations argue against the involvement of PTP in the release of cytochrome c. It was shown that cyclosporin A does not block Bax- or Bid-induced cytochrome c release from mitochondria (Bossy-Wetzel et aI, 1998). Cytochrome c release occurs before a drop in mitochondrial membrane potential which reflects PTP opening (Mancini et aI, 1997) and during apoptosi s in several cell types, mitochondria do not swell but rather shrink, a process described as "mitochondrial pyknosis" (Anjum et aI, 2001) . Our studies on apoptosis in tumor cells have demonstrated cytochrome c release to be independent of caspases and Bcl-2 overexpression (Muchmore et aI , 1996).

(ii) The second model envisages the formation of a cytochrome c conducting channel in the outer mitochondrial membrane regulated by Bcl-2 family of proteins. The clue that Bcl-2 members form channels came from the three dimensional structure of Bel-XL and Bid, which resembles the structure of pore­forming domains of diptheria toxin and some bacterial colicins (Schlessinger et aI, 1997). A likely candidate for the formation of this channel is the pro-apoptotic protein, Bax. It was shown that an oligomer of Bax

can form large-conductance channels in lipid bilayers (Antonsson et aI, 1997). Moreover, addition of Bax directly to isolated mitochondria triggers release of cytochrome c through a mechanism insensitive to PT blockers (B ossy-Wetzel et ai, 1998) .

Yet another model involves Bax cooperating with VDAC to fo rm a cytochrome c-conducting channel (Gross et ai, 1999). Following binding of Bax to the VDAC, the confo rmation of :he VDAC would change, leading to the formation of a channel that is permeable to cytochrome c. But as of now, there is no evidence that Bel-2 family proteins fo rm channels in vivo.

Regulation of Mitochondrial Homeostasis and Cell Death by Bcl-2 family of Proteins

The Bel-2 family of proteins constitute one of the most crucial classes of apoptosis regulatory proteins. These inelude both the pro-apoptotic (Bax , Bad, Bak, Bid, Bcl-Xs, Bik, Bim) and anti-apoptotic (Bel-2, Bel­XL Bclw, Mcl,) proteins (Kroemer, 1997). These proteins are mostly localized to the outer membranes of mitochondria, endoplasmic reticu lum and nueleus, as a result of a carboxy terminal membrane anchor (Hsu et aI, 1997). Some of them are cytosolic (Bax , Bel-XL, Bid, Bad and Bim) but translocate to mitochondria during apoptosis (Desagher et ai, 1999). Members of this family are capable of dimerization and the ratio of pro- to anti-apoptotic molecules determine, in part, the susceptibility of cells to undergo programmed cell death (Vander Heiden et aI, 1997). These proteins regulate apoptosis in part by affecting the mitochondrial compartmentalization of cytochrome c. Expression of Bcl-2 or Bel-XL prevents the redistribution of cytochrome c in response to multiple death inducing stimuli (Yang et ai, 1997; Manon et aI, 1997), while Bax promotes cytochrome c release (Rosse et al 1998). Bcl-2 expression inhibits the generation of reactive oxygen species (Kane et ai , 1993) and intracellular acidification and stabilizes the mitochondrial membrane potential (Manon et ai, 1997; Anjum & Khar, 2001) . Bel-2 can also effect mitochondrial proton flux (Zhu et aI, 1999) and modulate calcium homeostasis (Vaux & Strasser, 1996).

Mitochondria-dependent and -independent Pathways In some type of cells, apoptosis triggered by Fas

(CD9S) and certain members of the TNF family of death receptors is not blocked by over-expression of Bel-2 or other anti-apoptotic members of the Bcl-2

66 INDIAN J BIOTECHNOL, JANUARY 2002

family, circumventing the participation of mitochondria or other organelles, where Bcl-2 and many of its homologues reside as integral membrane proteins (type I cells) (Kuwana et ai, 1998),

However in some cells, Bcl-2 is capable of suppressing Fas- or TNF-induced apoptosis (type II cells). The basis for this difference in Bcl-2 sensitivity appears to reside in whether caspase-8 does or does not require a mitochondria-dependent amplification step to achieve sufficient activation of downstream effector caspases for apoptosis. Addition of caspase-8 at high concentration to cytosolic extracts results in downstream caspase activation and apoptosis, whereas at lower concentrations of caspase-8, the downstream caspase activation is minimal, unless mitochondria are added to cytosolic extracts (Cleveland & Ihle, 1995).

The Fas and TNF-a Signal Transduction-A Prototype of Cell Death Pathway

Fas (CD95) and TNF receptor 1 (TNFRl) are cell surface receptors that when trimerized with ligand (FasL and TNF-a respectively), induce apoptosis (Nagata & Goldstein, 1995). These pathways playa critical role in regulation of the immune system. Inappropriate expression of the FasL can enable tumor cells to escape immune surveillance (Khar et ai, 1998; Griffith et ai, 1995) and expression is usually restricted to sites of immune privilege.

Apoptotic singling by Fas and TNFRI is mediated by a stretch of 80 amino acids, called the death domain (DD) in the cytoplasmic portion of each receptor. Adaptor proteins (FADD I Mort 1, RIP and T RADD) bind to these DDs via their own DDs (Chinnaiyan et ai, 1995; Kischkel et ai, 1995). Over­expression of FADD and RIP causes apoptosis. FADD also contains a motif at its amino terminus, the death effector domain (DED), which binds to the pro­domain of caspase-8. A complex of proteins are found associated with stimulated CD95 (Designated as CAP 1-4) (Thome et ai, 1997). Together with CD95 and caspase-8, these proteins form a complex called the death-inducing signaling complex (DISC). CAP4 contain two DEDs at its N-terminus and showed the typical domain structure of an ICE-like protease at its C-terminus. It was therefore termed FLICE (for FADD-like ICE), now called caspase-8 (Tewari et ai, 1995). After stimulation FADD I Mort 1 and caspase-8 are recruited to CD95 within seconds after receptor engagement. Direct binding of caspase-8 causes

structural changes in the molecule that result in the autoproteolytic activation . The active subunits pIO and p18 are released into the cytoplasm. The active caspase-8 cleaves various cellular death substrates, including other caspases, such as caspase-3, thus initiating the execution of apoptosis .

There are other DED containing proteins, some virally encoded, called the v-FLIPS (for viral FLICE inhibitory proteins) . vFLIPs consist of two DEDs and biochemical analysis of v-FLIP-transfected cells showed that they bind to the CD95/FADD complex and thus inhibit the recruitment of caspase-8 and a functional DISC formation (Wiley et ai, 1995).

TRAIL (APO-2L) Apoptosis System TRAIL or TNF-related apoptosis-inducing ligand

was identified on the basis of sequence homology to the other members of the TNF family (Degli-Esposti, 1999). TRAIL was found to induce apoptosis more efficiently in tumor cells than in normal cells. TRAIL can bind two apoptosis-inducing receptors, TRAIL­Rl (DR4) and TRAIL-R2 (Killer, DR5, TRICK2), two additional cell-bound receptors, incapable of transmitting an apoptotic signal, TRAIL-R3 and TRAIL-R4 (Walczak & Krammer, 2000). It was earlier thought that the existence of these functionally distinct receptors might provide an answer to the differential sensitivity to TRAIL, observed between normal and transformed cells, which was later disproved (Sedger et ai, 1999).

The physiological significance of this novel apoptosis-inducing system remained unknown for a long time. Interestingly, functional surface expression of TRAIL was often associated with stimulation by interferons. Therefore, it is likely that the antitumoral effect of IFNs may at least be partially mediated by TRAIL-induced direct killing of TRAIL-sensitive tumor cells (Bonavida et ai, 1999). A number of studies have been performed combining TRAIL with different chemotherapeutic drugs for the treatment of various malignancies (Kastan et ai, 1992). Most chemotherapeutic drugs and radiation therapy used in the treatment of malignancies lead to apoptosis primarily by engagement of the mitochondrial proapoptotic machinery. TRAIL induces apoptosis via a caspase signaling cascade that executes apoptosis independently of the proapoptotic machinery of mitochondria. TRAIL can bypass the anti-apoptotic effect of Bcl-2 or Bcl-XL overexpression. TRAIL and chemotherapeutic drugs work via distinct apoptotic

ANJUM AND KHAR: APOPTOSIS-THE CELL DEATH PROCESS 67

pathways and such a combinatorial treatment of cancer may diminish the chances of the tumour to develop further.

Apoptosis and p53 The tumor suppressor protein p53 plays mUltiple

roles in cells. Expression of high levels of wild type (but not mutant) p53 has two outcomes: cell cycle arrest and apoptosis. Both provide mechanisms by which p53 functions to control DNA damage, protecting cellular descendants from accumulating excessive mutations. In response to irradiation or other DNA-damaging insults, p53 levels rise and produce G 1 arrest (Pines, 1994). Possible mechanisms include p53-mediated transcription of genes such as p21/Waf/Cip, which inhibit cyclinlcdk activity and thereby inhibit cyclin-dependent entry into S phase, as well as DNA replication machinery (Clarke et ai, 1993).

p53 mediates apoptosis in several cell types induced by different stimuli such as DNA damage (Deb bas & White, 1993) adenovirus EIA expression (Hermeking & Eick, 1994), myc expression (Johnson et ai, 1993) or withdrawal of growth factors (Lowe et ai, 1993). However, apoptosis can also occur by p53-independent pathways (Debbas & White, 1993; Lowe et ai, 1994). The p53-dependent apoptotic pathway has been shown to be an important mechanism by which transformation is suppressed in oncogene­expressing cells (Yuan & Horvitz, 1992).

Programmed Cell Death in Invertebrates (i) peD in C. elegans

The existence of an intrinsic cell suicide program was ascertained through genetic studies in the nematode C. elegans that identified genes involved in the cell death program and its control (Ellis & Horvitz, 1986). The genes CED-3 and CED-4 (the homologues of ICE and Apaf-l in mammals) are required for execution of cell death and CED-9 (homologue of Bcl-2 in mammals) inhibits cell death in C. elegans (Hengartner & Horvitz, 1994; Chinnaiyan et ai, 1997). Mutational analysis of these genes in C. elegans have defined a sequential death pathway. Using yeast-two hybrid analysis it was demonstrated that CED-4 can bind to CED-9, CED-3 or both simultaneously (Spector et ai, 1997) analogous to the mammalian 'apoptosome' consisting of Apaf-l, Bcl-XL and caspase-9 (White et ai, 1994), highlighting the functional similarity of the worm and human cell death machinery.

(ii) peD in Drosophila Many death regulatory genes have been identified

in Drosophila through mutation analysis. The first global cell death-defective Drosophila mutation was discovered following apoptosis in living fly embryos (Grether et ai, 1995). This rare phenotype was mapped to a complex locus, the Reaper region. Later three cell-death activators were discovered Reaper (Rpr) , Grim and Hid which mapped to the same locus (Grether et ai, 1995; Dorstyn et ai, 1999). Cell death induced by each of these proteins are preceded by caspase activation and inhibited by caspase inhibitors. Caspases characterized in flies include Dredd (Dorstyn et ai, 1999) and Dronc (Song et al 1997), which resemble effector caspases and DCP-l (Fraser & Evan, 1997) and Drice (Rodriguez et ai, 1999) resembling executioner caspases. Dark-a fl y homologue of Apaf-lICED-4 relays death signals to apoptotic caspases (Greenberg, 1996).

(iii) peD in Plants, Fungi and Bacteria Cell death associated with the hypersensitive

response (HR) in several plant-pathogen interactions has morphological similarities to mammalian apop­tosis (Mittler & Lam, 1996). Caspase-like proteolytic activity was detected in tobacco plant tissues that were developing HR following infection with tobacco mosaic virus (TMV) (JUrgens meier et ai, 1997). In addition, some types of plant cell death are accompanied by DNA damage often with the characteristics of endonucleotically processed DNA, one of the hallmarks of apoptosis (Ellis & Horvitz, 1986).

The pro-apoptotic proteins Bax and Bak are capable of provoking death of the yeast Saccharo­myces pombe (Greenhalf et ai , 1996). The expression of Bax in S. cerevisiae is lethal in the presence of a functional respiratory chain and Bcl-2 inhibited yeast cell death (Bishop et ai, 1998). Furthermore, mito­chondri a of Bax-expressing yeast cells release cytochrome c which is inhibited by coexpression of Bcl-XL (Rosse et ai , 1998) . These observations suggest that some elements involved in mammalian cell death exist in yeasts.

The best characterized programmed cell death mechanism of bacteria are found under starvation conditions where moribund cells are programmed to release their contents by lysis, thereby providing nutrients to the remaining healthy cells in the population, termed GASP (growth advantage in

68 INDIAN J BIOTECHNOL, JANUARY 2002

stationary phase). The 'entericidin locus' of E. coli governs stationary phase bacteriolysis (Hickman, 1992).

De-regulation of Apoptosis and Disease Diseases characterized by accumulation of cells

include cancer, autoimmune and certain viral diseases. Cell accumulation can result from either increased proliferation or the failure of cells to undergo apoptosis in response to appropriate stimuli (Thompson, 1995).

Apoptosis is a protective measure preventing malignant transformation. In this context, organisms may have evolved an efficient mechanism to eliminate cells that have sustained genetic lesions resulting in unscheduled division, provided that such transformed cells are triggered to undergo apoptosis in response to adverse growth signals. Effective tumour therapy may involve the induction of apoptosis in tumour cells rather than inhibition of cell proliferation. DNA damage induced by radiation and drug treatment in combination therapy leads to apoptotic cell death (Cohen, 1991). Earlier studies have revealed that the lymphocyte-induced killing of tumour targets is associated with the fragmentation of target cell DNA (Cohen, 1991). In vivo and in vitro studies have demonstrated the occurrence of DNA fragmentation in the NK cell-mediated spontaneous regression of AK-5, a macrophage tumour (Kausalya et ai, 1994; Bright et ai, 1994).

Excessive cell death can result from acquired or genetic conditions that enhance the accumulation of signals that induce apoptosis or that decrease the threshold at which such events induce apoptosis. These include virus-induced lymphocyte depletion in AIDS, cell death in neuro-degenerative disorders like Alzheimer's disease, Parkinson's disease, retinitis pigmentosa and various forms of cerebral degeneration (Thompson, 1995).

Acknowledgement Authors thank Mrs. T. Hemalatha for excellent

secretarial assistance. Financial support was provided by the Council of Scientific & Industrial Research and Department of Biotechnology, Government of India.

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