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Apoptotic cell death : when death is a useful process.
For nearly two decades, studies in cancer research focussed on identifying genes that act as positive and negative regulators of cell growth. The understanding that a programmed pattern may control cell death, as is known to be the case for cell differentiation and proliferation, is a relatively recent development in biology , as is the recognition that the regulation of cell death is also an important modulator in the predisposition to tumorigenesis .
Kerr and colleagues  who used morphological criteria to define apoptosis as a specialized form of cell death distinct from the cellular breakdown seen in lytic cell death, also known as necrosis or unscheduled accidental cell death.
Research in this field is complicated, at least partly by the fact that everything from oncoproteins, tumour suppressor proteins, growth factors to signaling pathways seems able to induce apoptosis and everything seems to be able to suppress apoptosis .
Features of apoptosis
Apoptosis proceeds in four phases : the initition phase, the detection phase, the effector phase with the fourth phase of cell death being the postmortem or degradative phase, the vital structures and functions are destroyed giving rise to alterations commonly used to define and identify this type of cell death .
Apoptosis (from the Greek apo : off and ptosis : a falling off or dropping off) is defined as ‘a single deletion of scattered cells by fragmentation into membrane-bound particles which are phagocytosed by other cells; believed to be due to programmed cell death’ (Stedman’s Medical Dictionary, 1995) .
Apoptotic cells have specific biochemistry and morphology and the most rigorous standards for definition of apoptosis remain chromatin condensation, cytoplasmic vacuolization and plasma membrane blebbing .
Typically, cells shrink and display surface alterations such as widespread membrane blebbing and exposure of molecules normally confined to the interior of the cell such as exposure of phosphatidylserine on the outer surface of the plasma membrane. Blebbing, chromatin condensation and cell shrinkage occur in parallel but the integrity of the cell membrane is preserved much longer than in necrosis.
The cells also undergo a series of typical nuclear changes such as chromatin condensation and marginalisation where the chromatin condenses and aggregates along the nuclear membrane in a crescent-shape pattern .
Chromatin condensation is accompanied by DNA cleavage which has been shown to involve three discrete stages: initially, to fragments of approximately 300 kb, followed by the appearance of 50 kb fragments, and, finally, formation of small oligonucleosomal-sized fragments due to activation of a Ca2+, Mg2+-nuclease and internucleosomal fragmentation [1,3,7]. When separated by electrophoresis, these fragments form a typical ‘ladder’ which is however not always observed in spite of a clear apoptotic morphology .
The cell rounds up, dissociates itself from its neighbours and shrinks dramatically, throwing out protuberances or blebs that eventually separate into the membrane-bound chromatin containing "apoptotic" bodies without lysis [4,9].
Early in the process of apoptosis, the plasma membrane expresses pro-phagocytic signals such as phosphatidylserine which flips from the inner to the outer lipid bilayer and this is recognised by receptors for these phospholipids on the surface of phagocytes. This is relevant for the second stage, in vivo, where the dying cells and apoptotic bodies are recognized and engulfed by other cells in a clearance phase [9,10] where they undergo a series of changes resembling in vitro autolysis within phagosomes, and are rapidly degraded by lysosomal enzymes derived from the ingesting cells . The sequestration of apoptotic cells by professional phagocytes, due to display on the outer surface of molecular components which are normally masked or restricted to the cell interior, before they can lyse, spill their contents and cause an inflammatory reaction, is a physiological advantage .
In vitro at least, one of the latest stage of this degradative phase appears to be the loss of membrane integrity, secondary necrosis, which occurs after DNA degradation .
Genetic Regulation of apoptosis
The proper execution phase of cell death appears to be controlled by the elements of a core genetic program which seems to be constitutively expressed in virtually every cell . The expression of more than 30 genes coincides with programmed cell death and these include transcription factors, proteases and protease inhibitors .
Among the most widely studied genes involved in apoptosis have been the antiapoptotic and pro-apoptotic bcl-2 family and the tumour-suppressor p53 gene [2,8,11].
Inappropriate expression of the normal bcl-2 protein appears to be a cause of tumour formation in cases studied  and overexpression of the antiapoptotic gene bcl-2 leads to lymphoid hyperplasia, lymphomas and self-agression by self-reactive lymphocytes normally deleted via apoptosis. On the other hand, mice lacking bcl-2 lose their lymphoid system in postnatal life due to an abnormal susceptibility to undergo apoptosis .
High levels of bcl-2 expression has been recently associated with poor outcome of chemotherapy in acute myeloid leukaemia [13,14] and resistance to apoptosis provided by bcl-2 expression extends across a spectrum of agents [15,16].
Besides bcl-2 family, another family of antiapoptotic proteins receiving increasing attention are the inhibitor of apoptosis proteins (IAP) . Another family of proteins that has advanced the understanding of the molecular basis of programmed cell death (PCD) in animal cells are the caspases, a family of proteases .
Functions of apoptosis
All multicellular organisms have mechanisms for killing their own cells, and use physiological cell death for defence, normal development of an organism such as embryonic or fetal development, in cell turnover in normal adult tissue (homeostasis) and aging .
Cell death is required for the removal for various types of cells [19,20] including : (1) cells that appear to have no function, (2) cells that are generated in excess, (3) cells that develop improperly, (4) cells that have completed their functions and (5) cells that are harmful.
Apoptosis also occurs spontaneously in untreated malignant neoplasms . It is implicated in both physiological involution (tissue regression) and atrophy of various tissues and organs. It can also be triggered by noxious agents .
Apoptosis and disease
Failure to undergo apoptosis or insufficient apoptosis results in malformation, chronic inflammatory diseases and autoimmune disorders as well as carcinogenesis, including lymphoproliferative disorders [2,9,11]. It is thus possible that malignancy may result from too few cells dying rather than too few cells being born . On the other hand, increased apoptosis is thought to contribute to neurodegenerative diseases and even AIDS .
Apoptosis and Cancer Therapy
A new perspective on anticancer therapeutic efficacy and the possiblity of new targets for drug action is offered by the principle that cytotoxic chemotherapeutic drugs operate primarily by causing apoptosis in tumour cells and participate in tumour regression and it has been suggested that a clearer understanding of this process will result in better cancer therapy [8,9,23-26]. Apoptosis occurs in many patients after antileukaemic chemotherapy as evidenced by DNA fragmentation detected in leukaemic blasts harvested from the bone marrow or peripheral blood of patients after treatment with a variety of antileukaemic agents .
This finding has prompted an investigation of anti-neoplastic therapies that more directly target the abberant control of apoptosis in tumours  and the biochemical events involved in apoptosis have been explored with respect to their relevance in cancer chemotherapy .
Laboratory study of Apoptosis
With the realization of the importance of this process, a variety of techniques have been used to study apoptosis and the principle underlying these are detailed below .
A study of morphological changes remains the most reliable method for identifying and quantifying apoptosis  and it has been suggested that as a rule, classification of cell death in a given model should always include morphological examination coupled with at least one other assay .
As a means of identifying apoptotic cell populations, detection of DNA ladders has been second only to morphological assessment in its applicability and utility . However, a number of problems are associated with this technique. Apoptosis without concommittant DNA fragmentation has been reported [30-34].
Moreover, the concept of DNA fragmentation as a marker of apoptosis has been seriously questioned as it was found to occur in necrotic cells too [35-38] or concomittant with indicators of cell lysis [39-41]. Thus, internucleosomal DNA fragmentation can no longer be used as a sole criterion for the identification of apoptosis .
High molecular weight (50 kb-600 kb) DNA fragmentation assessed by pulse-field gel electrophoresis is now a prefered marker of apoptotic DNA fragmentation and may occur in the absence of internucleosomal fragmentation [8,9,43].
Flow cytometry has also been used to detect DNA fragmentation . DNA fragmentation results in leakage of DNA out of the cell giving rise to a population of cells with a subdiploid DNA content which takes up less propidium iodide stain and appears as a sub G1 peak in the DNA histogram. This technique however does not allow differentiation between apoptotic and necrotic DNA degradation.
Other techniques include annexin V staining which binds to phosphatidylserine exposed early on the surface of apoptotic cells .
Loss of plasma membrane integrity
The loss of plasma membrane integrity is an event which occurs early in necrosis and late in apoptosis . It has thus been proposed that delayed loss of membrane permeability rather than DNA fragmentation be used as an indicator of apoptosis [8,44].
More recently enzyme assays have started to be used to study enzymatic activities responsible for morphological and biochemical changes that occur during PCD. The enzymes targeted have involved mainly RNA and protein synthesis inhibitors, protease inhibitors and nuclease inhibitors .
Clinical usefulness of the identification of apoptosis
The process of apoptosis can be used to evaluate drug toxicity and therapeutic efficacy in the clinical setting . Care should however be taken in choosing the right apoptotic marker and it should be borne in mind that cells may be clinically dead before apoptotic markers are expressed [46,47].
Several factors probably contribute to an underestimation of the role of PCD after chemotherapy in the clinical setting . For instance, apoptotic cells are rapidly cleared by phagocytosis. In addition, the extension of the observations from in vitro systems to more complex in vivo conditions might be associated with discrepencies. Because apoptosis is a stochastic process that involves a small percentage of cells at any time, detection of apoptotic cancer cells after chemotherapy in vivo requires either fortuitous sampling (i.e at a time when a high percentage of cells are apoptotic) or performance of sequential biopsies which is a costly process, exception made for sampling of leukaemic cells which are accessible from the bloodstream or bone marrow.
To conclude, in spite of the many recent advances made, the understanding of the regulation of programmed cell death in health and disease is far from complete, and the challenge of converting that understanding into new and effective therapeutic and analytical modalities has only begun to be approached [9,23]. The ultimate challenge may be to translate the knowledge gained into treatment strategies to predict through laboratory tests and improve clinical outcome in the many diseases linked to disregulation of apoptosis.
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