Kerr, Wyllie, and Currie first used the term apoptosis in a paper in 1972 to describe a morphologically distinct form of cell death, although certain components of the apoptosis concept had been described years previously. Our understanding of the mechanisms involved in the process of apoptosis in mammalian cells transpired from the investigation of programmed cell death that occurs during the development of the nematode Caenorhabditis elegans (Horvitz, 1999). In this organism 1090 somatic cells are generated in the formation of the adult worm, of which 131 of these cells undergo apoptosis or “programmed cell death.”
These 131 cells die at particular points during the development process, which is invariant between worms, demonstrating the accuracy and control in this system. Apoptosis has been recognized and accepted as an important mode of “programmed” cell death, which involves the genetically determined elimination of cells. However, there is other forms of programmed cell death have been described and other forms of programmed cell death may yet be discovered
Apoptosis occurs normally during development and aging and as a homeostatic mechanism to maintain cell populations in tissues. Apoptosis also occurs as a defense mechanism such as in immune reactions or when disease or noxious agents damage cells. Although there are a wide variety of stimuli and conditions, both physiological and pathological, that can trigger apoptosis, not all cells will necessarily die in response to the same stimulus. Irradiation or drugs used for cancer chemotherapy results in DNA damage in some cells, which can lead to apoptotic death through a p53-dependent pathway. Some hormones, may lead to apoptotic death in some cells although other cells are unaffected or even stimulated.
Some cells express Fas or TNF receptors that can lead to apoptosis via ligand binding and protein cross-linking. Other cells have a default death pathway that must be blocked by a survival factor such as a hormone or growth factor. There is also the issue of distinguishing apoptosis from necrosis, two processes that can occur independently, sequentially, as well as simultaneously (Zeiss, 2003). In some cases it’s the type of stimuli and/or the degree of stimuli that determines if cells die by apoptosis or necrosis. At low doses, a variety of injurious stimuli such as heat, radiation, hypoxia and cytotoxic anticancer drugs can induce apoptosis but these same stimuli can result in necrosis at higher doses. Finally, apoptosis is a coordinated and often energy-dependent process that involves the activation of a group of cysteine proteases called “caspases” and a complex cascade of events that link the initiating stimuli to the final demise of the cell Loss of control of apoptosis may result in disease. Excessive apoptosis is implicated in AIDS and Alzheimers disease and insufficient apoptosis may lead to cancer.
Morphology of Apoptosis
Light and electron microscopy have identified the various morphological changes that occur during apoptosis. During the early process of apoptosis, cell shrinkage and pyknosis are visible by light microscopy. With cell shrinkage, the cells are smaller in size, the cytoplasm is dense and the organelles are more tightly packed. Pyknosis is the result of chromatin condensation. On examination with hematoxylin and eosin stain, apoptosis involves single cells or small clusters of cells. The apoptotic cell appears as a round/oval mass. Plasma membrane blebbing occurs followed by karyorrhexis and separation of cell fragments into apoptotic bodies during a process called “budding.” Apoptotic bodies consist of cytoplasm with tightly packed organelles with or without a nuclear fragment.
The organelle integrity is maintained and all of this is enclosed within an intact plasma membrane. These bodies are subsequently phagocytosed by macrophages, or neoplastic cells and degraded within phagolysosomes. Macrophages that engulf and digest apoptotic cells are called “tingible body macrophages” and are found within the germinal centers of lymphoid follicles or within the thymic cortex. There is no inflammatory reaction with the process of apoptosis nor with the removal of apoptotic cells because: (1) apoptotic cells do not release their cellular constituents into the surrounding interstitial tissue; (2) they are quickly phagocytosed by surrounding cells thus likely preventing secondary necrosis; and, (3) the engulfing cells do not produce anti-inflammatory cytokines.
Distinguishing Apoptosis from Necrosis
The alternative to apoptotic cell death is necrosis, which is considered to be a toxic process where the cell is a passive victim and follows an energy independent mode of death. Oncosis is used to describe a process that leads to necrosis with karyolysis and cell swelling whereas apoptosis leads to cell death with cell shrinkage, pyknosis, and karyorrhexis.
Although the mechanisms and morphologies of apoptosis and necrosis differ, there is overlap between these two processes. Necrosis and apoptosis represent morphologic expressions of a shared biochemical network described as the “apoptosis-necrosis continuum” .For example, two factors that will convert an ongoing apoptotic process into a necrotic process include a decrease in the availability of caspases and intracellular ATP Whether a cell dies by necrosis or apoptosis depends in part on the nature of the cell death signal, the tissue type, the developmental stage of the tissue and the physiologic milieu (Zeiss, 2003).
It is not always easy to distinguish apoptosis from necrosis, they can occur simultaneously depending on factors such as the intensity and duration of there stimulus, the extent of ATP depletion and the availability of caspases (Zeiss, 2003). Necrosis is an uncontrolled and passive process that usually affects large fields of cells whereas apoptosis is controlled and energy-dependent and can affect individual or clusters of cells. Necrosis is caused by factors external to the cell or tissue, such as infection, toxins, or trauma that result unregulated digestion of cell components
Some of the major morphological changes that occur with necrosis include cell swelling; formation of cytoplasmic vacuoles; distended endoplasmic reticulum; formation of cytoplasmic blebs; condensed, swollen or ruptured mitochondria; disaggregation and detachment of ribosomes; disrupted organelle membranes; swollen and ruptured lysosomes; and eventually disruption of the cell membrane. This loss of cell membrane results in the release of the cytoplasmic contents into the surrounding tissue, sending chemotatic signals with eventual recruitment of inflammatory cells. Because apoptotic cells do not release their cellular constituents into the surrounding tissue and are quickly phagocytosed by macrophages or normal cells, there is essentially no inflammatory reaction. It is also important to note that pyknosis and karyorrhexis are not exclusive to apoptosis (Kurosaka et al., 2003).
Mechanisms of Apoptosis
The mechanisms of apoptosis are highly complex involving an energy dependent cascade of molecular events. Research indicates that there are two main apoptotic pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. However, there is now evidence that the two pathways are linked and that molecules in one pathway can influence the other. There is an additional pathway that involves T-cell mediated cytotoxicity and perforin-granzyme dependent killing of the cell. The perforin/granzyme pathway can induce apoptosis via either granzyme B or granzyme A. The extrinsic, intrinsic, and granzyme B pathways converge on the same execution pathway. This pathway is initiated by the cleavage of caspase-3 and results in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, crosslinking of proteins, formation of apoptotic bodies, expression of ligands for phagocytic cell receptors and finally uptake by phagocytic cells.
Caspases have proteolytic activity and are able to cleave proteins at aspartic acid residues, although different caspases have different specificities involving recognition of neighboring amino acids. Once caspases are initially activated, there seems to be an irreversible commitment towards cell death. To date, ten major caspases have been identified and broadly categorized into initiators (caspase-2,-8,-9,-10), effectors or executioners (caspase-3,-6,-7) and inflammatory caspases (caspase-1,-4,-5). Caspase-11, which is reported to regulate apoptosis and cytokine maturation during septic shock, caspase-14, which is highly expressed in embryonic tissues but not in adult tissues .
Extensive protein cross-linking is another characteristic of apoptotic cells and is achieved through the expression and activation of tissue transglutaminase. Another feature is the expression of cell surface markers that result in the early phagocytic recognition of apoptotic cells by adjacent cells, permitting quick phagocytosis with minimal compromise to the surrounding tissue. This is achieved by the movement of the normal inward-facing phosphatidylserine of the cell’s lipid bilayer to expression on the outer layers of the plasma membrane. Externalization of phosphatidylserine is a well-known recognition ligand for phagocytes on the surface of the apoptotic cell.
Extrinsic Pathway—The extrinsic signaling pathways that initiate apoptosis involve transmembrane receptor-mediated interactions. These involve death receptors that are members of the tumor necrosis factor (TNF) receptor gene superfamily. Members of the TNF receptor family share similar cyteine-rich extracellular domains and have a cytoplasmic domain of about 80 amino acids called the “death domain”. This death domain plays a critical role in transmitting the death signal from the cell surface to the intracellular signaling pathways.The sequence of events that define the extrinsic phase of apoptosis are best characterized with the FasL/FasR and TNF-α/TNFR1 models. In these models, there is clustering of receptors and binding with the homologous trimeric ligand.
Upon ligand binding, cytoplasmic adapter proteins are recruited which exhibit corresponding death domains that bind with the receptors. The binding of Fas ligand to Fas receptor results in the binding of the adapter protein FADD and the binding of TNF ligand to TNF receptor results in the binding of the adapter protein TRADD with recruitment of FADD and RIP. FADD then associates with procaspase-8 via dimerization of the death effector domain.
At this point, a death-inducing signaling complex (DISC) is formed, resulting in the auto-catalytic activation of procaspase-8 . Once caspase-8 is activated, the execution phase of apoptosis is triggered. Death receptor mediated apoptosis can be inhibited by a protein called c-FLIP which will bind to FADD and caspase-8, rendering them ineffective. Another point of potential apoptosis regulation involves a protein called Toso, which shows to block Fas-induced apoptosis in T cells via inhibition of caspase-8 processing .
Intrinsic Pathway—The intrinsic signaling pathways that initiate apoptosis involve a diverse array of non-receptor-mediated stimuli that produce intracellular signals that act directly on targets within the cell and are mitochondrial-initiated events. The stimuli that initiate the intrinsic pathway produce intracellular signals that may act in either a positive or negative fashion. Negative signals involve the absence of certain growth factors, hormones and cytokines that can lead to failure of suppression of death programs, thereby triggering apoptosis. In other words, there is the withdrawal of factors, loss of apoptotic suppression, and subsequent activation of apoptosis.
Other stimuli that act in a positive fashion include, but are not limited to, radiation, toxins, hypoxia, hyperthermia, viral infections, and free radicals. All of these stimuli cause changes in the inner mitochondrial membrane that results in an opening of the mitochondrial permeability transition pore, loss of the mitochondrial transmembrane potential and release of two main groups of normally sequestered pro-apoptotic proteins from the intermembrane space into the cytosol. The first group consists of cytochrome c, Smac/DIABLO, and the serine protease HtrA2/Omi. These proteins activate the caspase dependent mitochondrial pathway. Cytochrome c binds and activates Apaf-1 as well as procaspase-9, forming an “apoptosome”
The clustering of procaspase-9 leads to caspase-9 activation. Smac/DIABLO and HtrA2/Omi are reported to promote apoptosis by inhibiting IAP activity. Additional mitochondrial proteins interact with and suppress the action of IAP
The second group of pro-apoptotic proteins, AIF, endonuclease G and CAD, are released from the mitochondria during apoptosis, but this is a late event that occurs after the cell has committed to die. AIF translocate to the nucleus and causes DNA fragmentation and condensation of peripheral nuclear chromatin. This early form of nuclear condensation is referred to as “stage I” condensation. Endonuclease G also translocates to the nucleus where it cleaves nuclear chromatin to produce oligonucleosomal DNA fragments.
AIF and endonuclease G both function in a caspase-independent manner. CAD is released from the mitochondria and translocates to the nucleus where, after cleavage by caspase-3, it leads to oligonucleosomal DNA fragmentation and a more pronounced and advanced chromatin condensation. This later and more pronounced chromatin condensation is referred to as “stage II”condensation
The control and regulation of these apoptotic mitochondrial events occurs through members of the Bcl-2 family of proteins .The tumor suppressor protein p53 has a critical role in regulation of the Bcl-2 family of proteins.The Bcl-2 family of proteins governs mitochondrial membrane permeability and can be either pro-apoptotic or antiapoptotic. 25 genes have been identified in the Bcl-2 family. Some of the anti-apoptotic proteins include Bcl-2, Bcl-x, Bcl-XL, Bcl-XS and some of the pro-apoptotic proteins include Bcl-10, Bax, Bad, Bim, and Blk. These proteins can determine if the cell commits to apoptosis or aborts the process. It is thought that the main mechanism of action of the Bcl-2 family of proteins is the regulation of cytochrome c release from the mitochondria.
Mitochondrial damage in the Fas pathway of apoptosis is mediated by the caspase-8 cleavage of Bid. This is one example of the “cross-talk” between the death-receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway. Serine phosphorylation of Bad is associated with 14-3-3, a member of a family of multifunctional phosphoserine binding molecules. When Bad is phosphorylated, it is trapped by 14-3-3 and sequestered in the cytosol but once Bad is unphosphorylated, it will translocate to the mitochondria to release cytochrome C.
Bad can also heterodimerize with Bcl-Xl or Bcl-2, neutralizing their protective effect and promoting cell death When not sequestered by Bad, both Bcl-2 and BclXl inhibit the release of cytochrome C from the mitochondria. Reports indicate that Bcl-2 and Bcl-XL inhibit apoptotic death primarily by controlling the activation of caspase proteases. An additional protein designated “Aven” appears to bind both Bcl-Xl and Apaf-1, thereby preventing activation of procaspase-9.
Puma and Noxa are two members of the Bcl2 family that are also involved in pro-apoptosis. Puma plays an important role in p53-mediated apoptosis. It was shown that, in vitro, overexpression of Puma is accompanied by increased BAX expression, BAX conformational change, translocation to the mitochondria, cytochrome c release and reduction in the mitochondrial membrane potential. Noxa is also a mediator of p53-induced apoptosis. Studies show that this protein can localize to the mitochondria and interact with anti-apoptotic Bcl-2 family members, resulting in the activation of caspase-9.
Caspase-3 is the most important of the executioner caspases and is activated by any of the initiator caspases (caspase-8, caspase-9, or caspase-10). Caspase-3 specifically activates the endonuclease CAD. In proliferating cells CAD is complexed with its inhibitor, ICAD. In apoptotic cells, activated caspase-3 cleaves ICAD to release CAD. CAD then degrades chromosomal DNA within the nuclei and causes chromatin condensation. Caspase-3 also induces cytoskeletal reorganization and disintegration of the cell into apoptotic bodies. Gelsolin, an actin binding protein, has been identified as one of the key substrates of activated caspase-3. Caspase-3 will cleave gelsolin and the cleaved fragments of gelsolin, in turn, cleave actin filaments in a calcium independent manner. This results in disruption of the cytoskeleton, intracellular transport, cell division, and signal transduction.
Phagocytic uptake of apoptotic cells is the last component of apoptosis. Phospholipid asymmetry and externalization of phosphatidylserine on the surface of apoptotic cells and their fragments is the hallmark of this phase. The mechanism of phosphatidylserine translocation to the outer leaflet of the cell during apoptosis has been associated with loss of aminophospholipid translocase activity and nonspecific flip-flop of phospholipids of various classes. Research indicates that Fas, caspase-8, and caspase-3 are involved in the regulation of phosphatidylserine externalization on oxidatively stressed erythrocytes however caspase-independent phosphatidylserine exposure occurs during apoptosis of primary T lymphocytes.
The appearance of phosphotidylserine on the outer leaflet of apoptotic cells then facilitates noninflammatory phagocytic recognition, allowing for their early uptake and disposal.This process of early and efficient uptake with no release of cellular constituents, results in no inflammatory response. (Fadok et al., 2001).
The process for apoptosis, is generally characterized by distinct morphological characteristics and energy-dependent biochemical mechanisms. Apoptosis is considered a vital component of various processes including normal cell turnover, proper development and functioning of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell death. Inappropriate apoptosis (either too little or too much) is a factor in many human conditions including neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. Excessive apoptosis results in diseases such as Alzheimer’s disease, Parkinson’s disease.
Cancer is an example where the normal mechanisms of cell cycle regulation are dysfunctional, with either an over proliferation of cells and/or decreased removal of cells. Tumor cells can acquire resistance to apoptosis by the expression of anti-apoptotic proteins such as Bcl-2 or by the down-regulation or mutation of pro-apoptotic proteins such as Bax. The expression of both Bcl-2 and Bax is regulated by the p53 tumor suppressor gene Alterations of various cell signaling pathways can result in dysregulation of apoptosis and lead to cancer.
The p53 tumor suppressor gene is a transcription factor that regulates the cell cycle and is the most widely mutated gene in human tumorigenesis. The critical role of p53 is evident by the fact that it is mutated in over 50% of all human cancers. p53 can activate DNA repair proteins when DNA has sustained damage, can hold the cell cycle at the G1/S regulation point on DNA damage recognition, and can initiate apoptosis if the DNA damage proves to be irreparable. Tumorigenesis can occur if this system goes awry. If the p53 gene is damaged, then tumor suppression is severely reduced. The p53 gene can be damaged by radiation, various chemicals, and viruses.