APOPTOSIS AND CELL DEATH (MECHANISMS, PHARMACOLOGY AND PROMISE FOR THE FUTURE)

Loss of cellular populations is a key limiting factor in many medically and socially high-impact diseases. Refinement of scientific technology in recent decades made detection of various forms of physiological cell death possible. Physiological cell death is very distinct from necrosis. It occurs in two distinct models, and perhaps a whole spectrum between these two. Classic apoptosis, in which most of the early morphological changes occur in the nucleus, and a lysosomal or cytoplasmic cell death, in which the early alterations take place in the cytoplasm (6,46). Apoptosis, a morphologically defined cellular death, is implicated in removal of cells during ontogenesis, physiological cellular turnover in adults, cytokine-induced tumor involution, clonal selection of T-lymphocytes, gerontopathology, neurodegenerative disorders, immunopathologic states, and even myocardial or cerebral ischemia (6,7,22,70). However, a loss of cell’s susceptibility to inducers of physiological cell death is likely an important component in development of malignancy and maintenance of drug resistance (4,68,69). Therefore, after years of academic ignorance, processes of programmed cell death have become a hot issue in scientific world.


Introduction
Loss of cellular populations is a key limiting factor in many medically and socially high-impact diseases. Refinement of scientific technology in recent decades made detection of various forms of physiological cell death possible. Physiological cell death is very distinct from necrosis. It occurs in two distinct models, and perhaps a whole spectrum between these two. Classic apoptosis, in which most of the early morphological changes occur in the nucleus, and a lysosomal or cytoplasmic cell death, in which the early alterations take place in the cytoplasm (6,46). Apoptosis, a morphologically defined cellular death, is implicated in removal of cells during ontogenesis, physiological cellular turnover in adults, cytokine-induced tumor involution, clonal selection of T-lymphocytes, gerontopathology, neurodegenerative disorders, immunopathologic states, and even myocardial or cerebral ischemia (6,7,22,70). However, a loss of cell's susceptibility to inducers of physiological cell death is likely an important component in development of malignancy and maintenance of drug resistance (4,68,69). Therefore, after years of academic ignorance, processes of programmed cell death have become a hot issue in scientific world.

Pathways of Cellular Demise
The term 'programmed cell death' appeared in 1960s in works on cell elimination in metamorphosing insect (30,31,50). This refers to a genetically invoked form of death (6). Morphologically it may assume a picture of either apoptosis or lysosomal cell death. Although morphological descriptions consistent with apoptosis have been present in literature since the 19th century (32), it has not been sooner than in 1970s, when Kerr and associates (22) unequivocally defined its histological appearance. Apoptosis serves as a one of three models of cell death during ontogenesis and its microscopical appearance is less marked than that of necrosis (6). It includes distinctive chromatin condensation, formation of cytoplasmic "finger-like" projections, which detach from the cell body and form apoptotic bodies, and heterophagocytosis of cellular remnants by surrounding tissue. Absence of inflammation, cellular edema and significant subcellular damage is characteristic, but is observed in necrosis. Histological appearance tends to be conserved with respect to type of cell and damage (65). Prompt phagocytosis as well as the absence of inflammatory reaction persuades pathologist to greatly underestimate the contribution of apoptosis in cell removal from population (6,7).
Cytoplasmic cell death may be seen in cells possessing large cytoplasm and is heralded by early and vast changes in cytoplasm. These involve increase in a number of lysosomes along with their redistribution, development of large autophagic vacuoles, and specific sequence of organelle removal. Very late in this process nuclear alterations similar to apoptosis take place (46). Energy resources probably remain generous until the very late stage.
Necrosis is opposed to physiological cell death (Tab. 1). It represents a morphological counterpart of energy resources loss, membrane penetrations, ruined control of ion flow and osmolysis resulting in uncontrolled loss of cellular content (14,61,62).
Using classical staining procedures it is almost impossible to quantify apoptosis due to a very short lifetime of morphologically evident apoptosis, which is believed to be at the level of tens of minutes. Classical microscopic analysis was the first method used to define apoptosis, and it should be kept in mind, that none of modern methods has replaced it. However, a myriad of new methods suitable for detection of certain apoptotic features has emerged, among them annexin V immunohistochemistry (66), multiparameter flow cytometry (11), TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining, and diphenylamine assay of DNA fragmentation. Apoptosis can be in most instances identified by characteristic breakdown of DNA into oligonucleosomal fragments, which give so called laddering appearance on electrophoresis (2). Pioneer observation of this feature should be probably granted to Czech researchers (55). Chemical asymmetry of plasma membrane is a characteristic feature of normal cells. However, early during apoptosis cells export phosphaditylserine residues normally confined to the inner leaflet of the plasma membrane to the outer leaflet, thus flagging apoptotic cell to phagocytes (35). This feature has been recently employed to identify apoptotic cells using immunolabeling techniques against annexin V, cell membrane confined phospholipid binding protein with a high affinity for phosphatidylserine (66).
Morphological manifestation of apoptosis is linked to its terminal stage. Only these latter stages of the whole process are heralded by cell rounding, cytoplasm blebbing, and nuclear condensation and fragmentation. Acquisition of typical apoptotic morphology is dependent on caspase-mediated and energy-dependent rearrangements of cytoskeleton.
Nuclear pyknosis and karyorrhexis are near-to-definite morphological features of apoptosis. Factors responsible for chromatin condensation and pyknosis include DNases, Acinus and AIF (Apoptosis Inducing Factor). Caspase-activated DNase (CAD) is a cytosolic protein inactivated by heterodimerization with its inhibitor ICAD. This heterodimer splits by action of caspase-3 on ICAD and CAD translocates into the nucleus, where it exerts typical internucleosomal chromatin cleavage (12). Acinus (apoptotic chromatin condensation inducer in the nucleus) is newly described chromatin-condensation factor involved in apoptosis. For full activation it requires double caspase cleavage and features an unique peculiarity as it exerts its chromatincondensing action without any detectable DNase activity (49). Both Acinus and CAD lead to histological appearance of karyorrhexis. Yet another factor, mitochondrial AIF, participates in nuclear changes. However, it produces large scale DNA fragmentation into pieces around 50 kb in length and gives a picture of peripheral chromatin condensation (58).
Caspases have been found to mediate cleavage of many cytoskeleton-associated proteins, among them Gas2 (3), gelsolin (25), and fodrin (35). Detachment of apoptotic cells from plate or from other tissue cells was found to be a consequence of calpain-mediated cleavage of cytoplasmic domain of integrin β3 subunit (40), which is required to maintain cellular adhesion and cytoskeletal association. On the other hand, studies employing microtubule-damaging drugs such vincristine suggest that microtubule damage is an important event in Bcl-2 inactivation via hyperphosphorylation and induction of apoptosis (57). This means that upstream intracellular mediators of apoptosis initiate cytoskeletal rearrangements, which in turn potenciate apoptotic cascade via inhibition of anti-apoptotic function of Bcl-2 oncoprotein. Moreover, initiation of apoptotic cascade at any point may cause self-amplification and inevitable cell death.
Interesting association between inhibition of apoptosis and a gain of metastatic capability was found in cells lacking expression of cytoskeleton-bound Death Associated Protein (DAP) kinase (20). Restoration of normal DAP kinase expression in high-metastatic tumor cells suppressed their metastatic ability. Links among suppression of apoptosis, cytoskeleton rearrangements, and neoplastic immortality and metastatic capability need further elucidation.
Susceptibility of cells to suicide varies significantly. Genetic control of apoptosis is mediated through several gene products. Some of them promote apoptosis (p53, TNF, Fas/CD95, bax, bak and bad), while the others (e.g. bcl-2, bcl-X L ) block apoptosis and promote cell survival (41,71).

Subcellular Mechanisms of Apoptosis
Apoptosis seems to be an old and conserved reaction based on a self-scarifying anti-viral defense originally deve- 64  loped in primitive eukaryotes. Execution of this process involves inhibition of protein synthesis at the level of translation initiation, proteolysis specifically involving degradation of DNA repair mechanisms, and polynucleotide degradation. This complex molecular signaling system includes feedback mechanisms tending toward activation of all elements of the execution platform if only one element is initially engaged (67). Because morphological alteration of nucleus was first observed in apoptotic elements, it was considered a coordinator of whole process. However, studies on enucleated cells have shown they can die with apoptosis as well (52). Although cellular suicide program is genetically encoded, its translation immediately before execution of apoptosis is not required. Apoptosis in certain cellular populations can be prevented with transcriptional and/or translational inhibitors (29,43,60). However, similar approach in different settings may provoke or accelerate apoptosis (5,10). The effector tool of apoptosis is a class of cysteine proteases localized in cytoplasm -caspases, also termed ICE-like enzymes (Interleukin-1β-Converting Enzyme) according to the first discovered member of this family. Two pathways of cell suicide exist. One triggered by signals created within the involved cell (unbalanced oxidative stress, calcium overload) and the other one initiated by signals generated from outside of the cell (TNF, Fas ligand, NO, glucocorticoids, actinomycin D). The intrinsic signals activate caspase-9, while extrinsic engages caspase-8. Both modes converge to sequential activation of other caspases, all process thus resembling limited proteolysis seen with blood clotting. Activated caspases digest structural proteins and degrade chromosomal DNA leading to death of the cell. Cell's decision to commit suicide is driven by death activators and is counterbalanced by the action of trophic factors, among them nerve growth factor, basic fibroblast growth factor, interleukin 2, insulin, and others.

Necrosis Apoptosis
Classical studies have been carried out in a worm Caenorhabditis elegans model, which features extensive removal of cell population during ontogenesis. Genes responsible for programmed cell death were identified as cell death genes (ced), and their corresponding proteins were termed CED. The genes ced-3 and ced-4 are essential for cell death; ced-9 antagonizes the activities of ced-3 and ced-4, thus resembling bcl-2 family in mammals. CED-3 is a counterpart of mammalian caspases, while the function of CED-4 resembles that of mammalian Apaf-1 (Apoptosis Protease-Activating Factor 1). In mammalian cells, Bcl-2 protein drags caspases to mitochondrial membrane and prevents their activation, and blocks release of cytochrome C, which is a potent activator of pro-caspase-3.
Indeed, recent studies provided evidence on rate limiting behavior of mitochondria in early stages of apoptosis (16,41). These have given the experimental basis for threestep model of apoptosis (58): 1. Premitochondrial phase -activation of apoptotic signaling pathways, including so-called upstream caspases (Fig. 1), 2. Mitochondrial phase -loss of mitochondrial inner membrane potential accompanied by a release of proteins activating apoptotic effectors (Fig. 1,2), 3. Postmitochondrial phase -activation and action of apoptotic effectors (catabolic proteases -downstream caspases, nucleases) leading to microscopical appearance of apoptosis (Fig. 1). Fig. 1: Sequence of main events after injurious stimulus leading to cellular death. Data published by Zamzami and associates (31,72) document, that lowering of mitochondrial transmembrane potential ∆Ψ m , and subsequent intramitochondrial production of reactive oxygen species are triggers of apoptosis. Apoptosis has been observed in those cells only, in which depolarization of inner mitochondrial membrane occurred.
It has been reported that disturbances of mitochondrial function and integrity paralleled by disruption of intracellular calcium homeostasis preceded apoptotic changes of nucleus (37,42). Mitochondrial calcium overload initiates opening of a special mitochondrial megachannel and is prerequisite of all further steps of apoptosis (31,42,44).
Loss of electrochemical gradient on inner mitochondrial membrane is mediated by a sudden increase in its permeability. This permeability transition creates a shunt for protons, lowers protonmotive force ∆Ψ m , and results in a cessation of mitochondrial ATP synthesis. In all likelihood, permeability transition is caused by opening of special proteinaceous multiple conductance channel, or "megapore" or "megachannel". This nonselective channel is probably permeable to any atomic ion as well as water and forms at the junction of inner and outer mitochondrial membranes (45). Its opening gives rise to massive ion movement accompanied by water with resulting edema and rupture of outer mitochondrial membrane. Intermembrane proteins capable of inducing caspases (cytochrome c and apoptosis inducing factor AIF) are thus released to cytosol (16,56,74). Moreover, caspases induce liberation of intermembrane proteins from other mitochondria (36), hence engaging in self perpetuating cycle leading to coordination of proapoptotic behavior among all mitochondria in a given cell. For that reason permeability transition of inner mitochondrial membrane takes on a chain reaction profile and spreads as outbreak affecting entire mitochondrial population (53). Loss of mitochondrial potential ∆Ψ m , is a common trait of necrosis and apoptosis (72). End result of the mitochondrial dysfunction leads to biological catastrophe culminating in disintegration of plasmatic membrane (necrosis), or to activation of apoptotic proteases with subsequent activation of endonucleases and manifestation of apoptosis. Cell's decision on which morphological presentation will be preferred depends on intensity of initiating factor and energetic charge of the cell (27,28,39). Cells low in energy undergo uncoordinated process of necrosis, yet cells with sufficient energy stores experience apoptosis. Other explanation may be that cells mainly dependent on anaerobic glycolysis (leukocytes) undergo apoptosis, while cells reliant on aerobic glycolysis tend to suffer from necrosis. This is consistent with our findings of necrotic neuronal death in our model of transient seven-minute global cerebral ischemia in dogs, where we repeatedly failed to morphologically identify apoptosis (15,47). However, mitochondrial damage consistent with apoptosis has been observed (Fig. 3).
Either preventive or postinsult application of immunomodulant agent cyclosporine A or tacrolimus (FK-506) in settings of disrupted blood-brain barrier protects neurons against apoptosis induced by ischemia-reperfusion injury (18,58,72). These compounds inhibit formation of mitochondrial megapore (54), similarly to performance of intracellular calcium chelators (63,64) and natural inhibitors of apoptosis -some of protein products of bcl-2 gene family. Employment of free radical scavengers can only retard ter- 66 minal phase of apoptosis -reduction of cellular volume (73), but not cellular death itself.
Immunosuppressant actions of cyclosporine A, tacrolimus and rapamycine are mediated by the drug binding intracellular target -immunophillin. Drug-immunofillin complex binds to and inhibits the phosphatase calcineurin, thus resulting in modification of special proteosynthesis. Immunophillin ligands, including nonimmunosuppressants that do not inhibit calcineurin, stimulate regrowth of damaged peripheral and central neurons (48). Furthermore, tacrolimus inhibits the activity of nitric oxide synthase. Nitric oxide is capable of inducing apoptosis via direct opening of mitochondrial megapore (19).
Cell death activators, both apoptotic and necrotic, may be identical (54). Cell's fate is likely to be defined by the intensity and duration of exposure to initiating event. This is supported by reported succession of necrosis and apoptosis in glutamate-induced model of excitotoxic neuronal death. Early survival of necrotic phase was determined by recovery of mitochondrial energy-producing machinery, and neurons surviving the necrotic phase underwent apoptotic transformation (1).

Conclusion
Known associations of apoptosis controlling and cellular growth gene mutations include familial adenomatous polyposis (APC gene), hereditary malignant melanoma (regulators of cyclin-dependent kinases), Lynch syndromes (microsatelit DNA mutations) and others (8). Mutations in the p53 tumor suppressor gene are amongst the most frequent genetic abnormalities identified in human solid neoplasms. Besides malignancy, downregulation of apoptosis controlling mechanism is seen with inability to handle some forms of chronic inflammation (26,51), e.g. ulcerative colitis and rheumatic arthritis. Upregulation of apoptosis has been implicated in many autoimmune, neurodegenerative, and ischemic disorders (21,23,67). Current immediate clinical applications of apoptosis-related research constitute estimation of anticancer chemotherapy effectiveness (33), survival prediction in acute leukemia (13), or reduction of allograft reperfusion injury after transplantation (24). Basic research has also recently questioned usage of lactated Ringer solution for acute shock therapy (9).
New knowledge of cellular death control is to be conceived by basic research. Thereafter, applied research should extend our pharmacological armamentarium with new approaches for therapy of ischemic disorders, malignant diseases, chronic inflammation and many others. Sufficient understanding of apoptosis and cell growth regulation yet requires more years of investigation. Nevertheless, new millenium may bring a significant breakthrough in treatment of many incurable and incapacitating diseases.