APOPTOSIS: The Gene Directed Programme of Cell Death
T.V. Anilkumar

Courtesy : Festschrift - Dr. S. Ramachandran

Introduction
Cell death is a common biological phenomenon, and in the animal body, all cells will have to die sooner (during the life of the organism) or later (at the time of death of the organism). Physiologists and embryologists have identified many types of cell death and they consider cell death as a programmed event. Programmed cell death, which occurs during the life of the animal, is essentially a physiological process. Cell death seen during organogenesis in an embryo, involution of hormone dependent organs in the adult tissues, e.g. thymus, elimination of auto-reactive lymphocytes in lymph nodes and shedding of cells from certain organs, e.g. superficial layers of skin epithelium, endometrium are some examples where cells die for physiological reasons. In these circumstances, it is not clear whether the cells are opting for a ‘cell suicide’ by themselves or they are killed by some microenvironmental factors such as neighbouring cells or some tissue factors. In any case, the cell death which occurs as a physiological process is generally called as ‘programmed cell death’ or ‘physiological cell death’. At least some forms of programmed cell death are genetically controlled and therefore many gene products are involved in initiation and execution of the programme for cell death. The morphology of cells dying by the gene directed programme of cell death has remarkable morphological resemblance to a certain type of cell death seen in some disease conditions. In contemporary biology this type of cell death with certain unique morphological features, detectable in either physiological or pathological settings is called ‘apoptosis’. 

The term ‘apoptosis’ (Greek, falling-off) was originally introduced into biology by a group of pathologists (1) in 1972 for describing a cell death type which they thought would be important in determining the doubling time of certain type of tumours. During the 70s, scientists did not consider apoptosis as a new biological entity, but the name caught the attention of mainstream biologists during mid 80s. Subsequently there was widespread confusion as well as controversy in defining apoptosis and assigning this nomenclature to different types of cell death in the animal body. In fact, the relationship and similarity of apoptosis with other forms of cell death are not clear and therefore many scientists use ‘cell suicide’, ‘physiological cell death’ and ‘programmed cell death’ as synonyms of apoptosis. The proper pronunciation of apoptosis is also controversial. It is now widely accepted that apoptosis does not represent all forms of ‘programmed cell death’ or physiological cell death, but it is considered as a common form of cell death in the animal body. Currently it is talked about as an important biological phenomenon with wide ranging implications, rather than just as a type of cell death. Indeed it is also a hot topic for research workers and over 50,000 publications have been made on the subject (2).

Morphology: apoptosis vs necrosis
The morphological differences between apoptosis and necrosis are well known (3). Necrosis is the most important form of cell death known to histo- pathologists, which could be appreciated in many disease conditions. In this type of cell death, cell swelling, rupture of cytoplasm, leakage of cell contents and initiation of inflammatory response in the surrounding tissue are consistently seen. On the other hand, apoptosis (Figs. 1 to 3) is characterised by nuclear chromatin condensation, cytoplasmic shrinking, dilated endoplasmic reticulum, and membrane blebbing. Mitochondria remain unchanged morphologically. The sequence of changes, ‘shrink me’, ‘blend me’, ‘crush me’ and ‘eat me’ are characteristic for apoptotic form of cell death, irrespective of the circumstances which have caused the death.

Apoptosis is often hard to observe in vivo because the dying cells are rapidly phagocytosed by tissue macrophages, and this phagocytosis is clearly different from that seen in inflammation, where activated macrophages are recruited from outside the immediate area of death. Any native cell, including non-activated macrophages can phagocytose an apoptotic body.

Biochemical mechanism
Scientists have yet to unravel the molecular mechanisms responsible for apoptosis. They have proposed a number of hypotheses from time to time, but none of them has been proved conclusively.

One of the most dramatic events in apoptosis is DNA fragmentation, which was described by Wyllie in 1980(4). When DNA from apoptotically dying cells was subjected to agarose gel electrophoresis, a ladder pattern was observed. The steps of the ladder represented DNA fragments of sizes corresponding to multiple integers of ~180 bp units, which is the length of inter-nucleosomal region of native chromatin. Whereas this is true in most forms of apoptotic cell death, DNA fragmentation is not uniform in all cases.

Apoptotic death can be triggered by a wide variety of intrinsic as well as extrinsic stimuli. The most studied death stimulus is DNA damage (5) (by irradiation or drugs used for cancer chemotherapy), which in many cells leads to apoptotic death via a pathway dependent on p53 protein. Some hormones such as corticosteroids also lead to this form of cell death in some cells e.g., thymocytes, and they are closely associated with the Bcl-2 super family of proteins (6). An important proved observation is that mitotic cells are prone to cell death by apoptosis. This also gives the impression that the apoptotic cell death and cell proliferation are closely linked events. Apoptosis could be a natural phenomenon to prevent the propagation of damaged DNA into progeny cells and cells with extensive damage opt for a suicide, i.e. better dead than wrong.

The changes in the apoptotic cells which trigger phagocytosis by resident cell population and non-activated macrophages have been investigated extensively. Macrophages appear to recognise apoptotic cells via several different recognition systems which seem to be used preferentially by different macrophage subpopulations. There is good evidence that apoptotic cells lose the normal phospholipid asymmetry in their plasma membrane as manifested by the exposure of normally inward- facing phosphatidyl serine on the external face of the bilayer. Macrophages can recognise this exposed lipid headgroup through an unknown receptor, triggering phagocytosis (7).

The studies on cell death abnormal genes (ced genes) in the nematode worm Caenorhabditis elegans, and identification of mammalian equivalents of these genes revolutionised the apoptotic research (8,9). A number of apoptotic and anti-apoptotic genes are now known: bcl-2, bax, and c-myc are some of them in the long list.

Another biochemical hallmark of apoptotic death, which increasingly appears general, is the activation of caspases (9-12). These are cysteine proteases and are widely expressed in an inactive proenzyme form in most cells. Their proteolytic activity is characterized by their unusual ability to cleave proteins at aspartic acid residues, although different caspases have different fine specificities involving recognition of neighbouring amino acids. Active caspases can often activate other pro-caspases, allowing initiation of a protease cascade. While several protein substrates have been shown to be cleaved by caspases during apoptotic death, the functionally important substrates are not yet clearly defined. The ability of specific caspase inhibitors to block cell death by apoptosis, as well as the demonstration of knockout mice lacking caspase 3, 8 and 9 fail to complete normal embryonic development supports the role of caspases in apoptosis. A critical issue is how caspases become initially activated, which seems to be an irreversible commitment towards death. It seems that aggregation of some pro-caspases with large pro- domains allow them to autoactivate. Clearly the role of caspases in causing apoptosis needs further examination.

Once it was thought that apoptosis represents an exclusive cell suicide phenomenon, implying that all apoptotic signals are intrinsic in nature. However, they are not always intrinsic in nature; many extrinsic stimuli could initiate this form of cell death. Some cell types express a surface protein, which initiates an intracellular death signal in response to crosslinking (13,14). These cell surface receptor molecules may play a pivotal role in initiating apoptosis. Much research is centred on CD95 (Fas/ Apo-1), a molecule which represents a superfamily of cell signalling molecules in the tumour necrosis factor (TNF) family. These are called as death receptors. It is believed that the messages from the cytoplasmic membrane are carried down into the cell, for activating certain signal transduction pathway and ultimately generate death signals internally. Often, ligand-crosslinking of death receptors may result in the formation of a cytoplasmic complex in which pro-caspase-8 is aggregated and activated.

Recent experiments demonstrate that mitochondria are involved in activation of pro-caspase-9. The interaction of cytochrome-c proteins of mitochondria and products of caspase activation results in ‘apoptosome’ a large protein complex (the little devil of death). Apoptosome may be responsible for regulation of fluid mechanics within the dying cell (15-18).

In short, a series of apoptotic and anti-apoptotic proteins are present in all cells. Apoptosis results after activation of a certain combination of some of these proteins and the grouping of proteins responsible for apoptosis is currently under debate. Many biochemical correlates have been postulated from time to time by several groups but these proposals require extensive elucidation, clarification and validation.

Significance of apoptosis
While there is much to be learned about the molecular pathways leading to apoptotic cell death, it is increasingly clear that cell death by apoptosis is a part of normal biological processes. Certainly our understanding of such death should be elucidated further. This would facilitate our ability to manipulate apoptosis in tissues. This could facilitate therapeutic intervention of many major diseases such as cancer, heart disease, stroke, AIDS, autoimmunity, degenerative diseases, and others (19). It is encouraging that some pharmaceutical preparations, capable of regulating apoptosis, are currently under clinical trials (20).
Apoptosis is vital in the resolution of inflammation since neutrophils undergo apoptosis and associated cell surface changes aid their phagocytosis by macrophages (6). Therefore apoptosis may be a major determinant of the outcome of diseases where inflammation is important. Not surprisingly apoptosis is increasingly described in infectious diseases. Viral infection can trigger host cell apoptosis (21), which can then limit virus production. On the other hand, the inhibition of apoptosis by viral genes will provide a selective advantage to the virus. Apoptosis could be a anti-viral mechanism but death of too many cells could be life threatening. Apoptosis is also a modulating factor in many bacterial, mycotic and protozoan infections.

Although apoptosis is perceived by some as exclusively a type of physiological cell death, a wide variety of noxious stimuli including anti-cancer drugs, hyperthermia, irradiation and environmental pollutants can cause apoptosis (22,23).

An important philosophical question yet to be answered is that whether apoptosis is a reversible process. Without hesitation one would agree that there is a point of no return after which it would be rather difficult to reverse this death process. There is increasing evidence that apoptosis is reversible so long as the apoptotic fragment has not been eaten-up by phagocytes. This is true at least in nematode worms (24). Therefore, there is a possibility that it could very well be exploited as a phenomenon of therapeutic interest.

Summary
Apoptosis is a process by which cells die in a controlled and programmed manner in response to specific stimuli often following extrinsic or intrinsic signals which ultimately cause the ‘switching on’ of cell death regulatory genes. Condensation of chromatin and cytoplasm, fragmentation of the cell and formation of membrane-bound bodies containing intact organelles (apoptotic bodies), and phagocytosis of these bodies by resident cells are the major morphological changes associated with apoptosis. Biochemically, activation of non-lysosomal endonuclease is a cardinal feature of this mode of cell death.

It is generally believed that a cascade of events is responsible for apoptosis and these are controlled and regulated at different check-points. Many scientists believe that the Bcl-2 family of proteins and the genes responsible for their synthesis play a major role in regulating this mode of cell death. The role of caspases is also debated in many forums. Recently, the mitochondial cytochrome-c has been shown to be involved in apoptotic mode of cell death. Whatever are the mechanisms within the cell, the signals responsible for initiating apoptosis could be extrinsic, which is mediated through certain cell surface receptors called Apo-1/Fas (identified as CD95) and signal transduction pathways.

Although our knowledge about the cellular and molecular mechanisms regulating apoptosis is bit hazy, it is an important mode of cell death and it is responsible for the pathogenesis of many diseases. There is hope that strategies for regulating apoptosis in tissues can be developed, which would ultimately be beneficial for therapeutic intervention of disease process. 

Authors Corresponding address: 

Dr. T.V. Anilkumar
Scientist, Veterinary Pathology,
Shree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura P.O., Trivandrum - 695 012, India

The views expressed in this article are of the author(s), and any clarifications can be obtained from the author(s).