German scientist Karl Vogt was first to describe the principle of apoptosis in In , anatomist Walther Flemming delivered a more precise description of the process of programmed cell death. However, it was not until that the topic was resurrected. While studying tissues using electron microscopy, John Foxton Ross Kerr at the University of Queensland was able to distinguish apoptosis from traumatic cell death. Currie , as well as Andrew Wyllie , who was Currie's graduate student,  at University of Aberdeen.
In , the trio published a seminal article in the British Journal of Cancer. Kerr, Wyllie and Currie credited James Cormack, a professor of Greek language at University of Aberdeen , with suggesting the term apoptosis. He shared the prize with Boston biologist H. Robert Horvitz. For many years, neither "apoptosis" nor "programmed cell death" was a highly cited term. Two discoveries brought cell death from obscurity to a major field of research: identification of components of the cell death control and effector mechanisms, and linkage of abnormalities in cell death to human disease, in particular cancer.
Sulston for their work identifying genes that control apoptosis. The genes were identified by studies in the nematode C. In Greek, apoptosis translates to the "falling off" of leaves from a tree. Hippocrates used the term to mean "the falling off of the bones". Galen extended its meaning to "the dropping of the scabs". Cormack was no doubt aware of this usage when he suggested the name. To show the derivation clearly, we propose that the stress should be on the penultimate syllable, the second half of the word being pronounced like "ptosis" with the "p" silent , which comes from the same root "to fall", and is already used to describe the drooping of the upper eyelid.
The initiation of apoptosis is tightly regulated by activation mechanisms, because once apoptosis has begun, it inevitably leads to the death of the cell. A cell initiates intracellular apoptotic signaling in response to a stress, which may bring about cell suicide. The binding of nuclear receptors by glucocorticoids ,  heat,  radiation,  nutrient deprivation,  viral infection,  hypoxia ,  increased intracellular concentration of free fatty acids  and increased intracellular calcium concentration,   for example, by damage to the membrane, can all trigger the release of intracellular apoptotic signals by a damaged cell.
A number of cellular components, such as poly ADP ribose polymerase , may also help regulate apoptosis. Before the actual process of cell death is precipitated by enzymes, apoptotic signals must cause regulatory proteins to initiate the apoptosis pathway. This step allows those signals to cause cell death, or the process to be stopped, should the cell no longer need to die.
Several proteins are involved, but two main methods of regulation have been identified: the targeting of mitochondria functionality,  or directly transducing the signal via adaptor proteins to the apoptotic mechanisms. An extrinsic pathway for initiation identified in several toxin studies is an increase in calcium concentration within a cell caused by drug activity, which also can cause apoptosis via a calcium binding protease calpain.
The mitochondria are essential to multicellular life. Without them, a cell ceases to respire aerobically and quickly dies. This fact forms the basis for some apoptotic pathways. Apoptotic proteins that target mitochondria affect them in different ways. They may cause mitochondrial swelling through the formation of membrane pores, or they may increase the permeability of the mitochondrial membrane and cause apoptotic effectors to leak out.
During apoptosis, cytochrome c is released from mitochondria through the actions of the proteins Bax and Bak. The apoptosome cleaves the pro-caspase to its active form of caspase-9 , which in turn activates the effector caspase Mitochondria also release proteins known as SMACs second mitochondria-derived activator of caspases into the cell's cytosol following the increase in permeability of the mitochondria membranes. SMAC binds to proteins that inhibit apoptosis IAPs thereby deactivating them, and preventing the IAPs from arresting the process and therefore allowing apoptosis to proceed.
IAP also normally suppresses the activity of a group of cysteine proteases called caspases ,  which carry out the degradation of the cell. Therefore, the actual degradation enzymes can be seen to be indirectly regulated by mitochondrial permeability. Two theories of the direct initiation of apoptotic mechanisms in mammals have been suggested: the TNF-induced tumor necrosis factor model and the Fas-Fas ligand -mediated model, both involving receptors of the TNF receptor TNFR family  coupled to extrinsic signals. TNF-alpha is a cytokine produced mainly by activated macrophages , and is the major extrinsic mediator of apoptosis.
FLIP inhibits the activation of caspase Apoptosis is known to be one of the primary mechanisms of targeted cancer therapy. In some types of cells type I , processed caspase-8 directly activates other members of the caspase family, and triggers the execution of apoptosis of the cell. In other types of cells type II , the Fas -DISC starts a feedback loop that spirals into increasing release of proapoptotic factors from mitochondria and the amplified activation of caspase This balance is the proportion of proapoptotic homodimers that form in the outer-membrane of the mitochondrion.
The proapoptotic homodimers are required to make the mitochondrial membrane permeable for the release of caspase activators such as cytochrome c and SMAC. Control of proapoptotic proteins under normal cell conditions of nonapoptotic cells is incompletely understood, but in general, Bax or Bak are activated by the activation of BH3-only proteins, part of the Bcl-2 family. Caspases play the central role in the transduction of ER apoptotic signals.
Caspases are proteins that are highly conserved, cysteine-dependent aspartate-specific proteases. There are two types of caspases: initiator caspases, caspase 2,8,9,10,11,12, and effector caspases, caspase 3,6,7. The activation of initiator caspases requires binding to specific oligomeric activator protein. Effector caspases are then activated by these active initiator caspases through proteolytic cleavage. The active effector caspases then proteolytically degrade a host of intracellular proteins to carry out the cell death program.
There also exists a caspase-independent apoptotic pathway that is mediated by AIF apoptosis-inducing factor. Amphibian frog Xenopus laevis serves as an ideal model system for the study of the mechanisms of apoptosis. In fact, iodine and thyroxine also stimulate the spectacular apoptosis of the cells of the larval gills, tail and fins in amphibians metamorphosis, and stimulate the evolution of their nervous system transforming the aquatic, vegetarian tadpole into the terrestrial, carnivorous frog. Negative regulation of apoptosis inhibits cell death signaling pathways, helping tumors to evade cell death and developing drug resistance.
Many pathways and signals lead to apoptosis, but these converge on a single mechanism that actually causes the death of the cell. After a cell receives stimulus, it undergoes organized degradation of cellular organelles by activated proteolytic caspases. In addition to the destruction of cellular organelles, mRNA is rapidly and globally degraded by a mechanism that is not yet fully characterized.
A cell undergoing apoptosis shows a series of characteristic morphological changes. Early alterations include:. Apoptosis progresses quickly and its products are quickly removed, making it difficult to detect or visualize on classical histology sections. During karyorrhexis, endonuclease activation leaves short DNA fragments, regularly spaced in size. These give a characteristic "laddered" appearance on agar gel after electrophoresis. Before the apoptotic cell is disposed of, there is a process of disassembly.
There are three recognized steps in apoptotic cell disassembly: . The removal of dead cells by neighboring phagocytic cells has been termed efferocytosis. Many knock-outs have been made in the apoptosis pathways to test the function of each of the proteins. In order to create a tumor necrosis factor TNF knockout, an exon containing the nucleotides — was removed from the gene. This exon encodes a portion of the mature TNF domain, as well as the leader sequence, which is a highly conserved region necessary for proper intracellular processing.
However, upon immunization with SRBC sheep red blood cells , these mice demonstrated a deficiency in the maturation of an antibody response; they were able to generate normal levels of IgM, but could not develop specific IgG levels. Apaf-1 is the protein that turns on caspase 9 by cleavage to begin the caspase cascade that leads to apoptosis. This assay is used to disrupt gene function by creating an intragenic gene fusion. When an APAF-1 gene trap is introduced into cells, many morphological changes occur, such as spina bifida, the persistence of interdigital webs, and open brain.
In addition, after embryonic day APAF-1 cells are protected from apoptosis stimuli such as irradiation. A BAX-1 knock-out mouse exhibits normal forebrain formation and a decreased programmed cell death in some neuronal populations and in the spinal cord, leading to an increase in motor neurons. The caspase proteins are integral parts of the apoptosis pathway, so it follows that knock-outs made have varying damaging results. A caspase 9 knock-out leads to a severe brain malformation. A caspase 8 knock-out leads to cardiac failure and thus embryonic lethality.
However, with the use of cre-lox technology, a caspase 8 knock-out has been created that exhibits an increase in peripheral T cells, an impaired T cell response, and a defect in neural tube closure. Finally, a caspase 3 knock-out was characterized by ectopic cell masses in the brain and abnormal apoptotic features such as membrane blebbing or nuclear fragmentation.
Distinguishing Necroptosis from Apoptosis
A remarkable feature of these KO mice is that they have a very restricted phenotype: Casp3, 9, APAF-1 KO mice have deformations of neural tissue and FADD and Casp 8 KO showed defective heart development, however in both types of KO other organs developed normally and some cell types were still sensitive to apoptotic stimuli suggesting that unknown proapoptotic pathways exist. In order to perform analysis of apoptotic versus necrotic necroptotic cells, one can do analysis of morphology by time-lapse microscopy , flow fluorocytometry , and transmission electron microscopy.
There are also various biochemical techniques for analysis of cell surface markers phosphatidylserine exposure versus cell permeability by flow cytometry , cellular markers such as DNA fragmentation  flow cytometry ,  caspase activation, Bid cleavage, and cytochrome c release Western blotting. It is important to know how primary and secondary necrotic cells can be distinguished by analysis of supernatant for caspases, HMGB1, and release of cytokeratin However, no distinct surface or biochemical markers of necrotic cell death have been identified yet, and only negative markers are available.
These include absence of apoptotic markers caspase activation, cytochrome c release, and oligonucleosomal DNA fragmentation and differential kinetics of cell death markers phosphatidylserine exposure and cell membrane permeabilization. A selection of techniques that can be used to distinguish apoptosis from necroptotic cells could be found in these references.
The many different types of apoptotic pathways contain a multitude of different biochemical components, many of them not yet understood. In a living organism, this can have disastrous effects, often in the form of disease or disorder. A discussion of every disease caused by modification of the various apoptotic pathways would be impractical, but the concept overlying each one is the same: The normal functioning of the pathway has been disrupted in such a way as to impair the ability of the cell to undergo normal apoptosis.
This results in a cell that lives past its "use-by-date" and is able to replicate and pass on any faulty machinery to its progeny, increasing the likelihood of the cell's becoming cancerous or diseased. A recently described example of this concept in action can be seen in the development of a lung cancer called NCI-H XIAPs bind to the processed form of caspase-9, and suppress the activity of apoptotic activator cytochrome c , therefore overexpression leads to a decrease in the amount of proapoptotic agonists.
As a consequence, the balance of anti-apoptotic and proapoptotic effectors is upset in favour of the former, and the damaged cells continue to replicate despite being directed to die. Defects in regulation of apoptosis in cancer cells occur often at the level of control of transcription factors. This degree of independence from external survival signals, can enable cancer metastasis. The tumor-suppressor protein p53 accumulates when DNA is damaged due to a chain of biochemical factors.
Part of this pathway includes alpha- interferon and beta-interferon, which induce transcription of the p53 gene, resulting in the increase of p53 protein level and enhancement of cancer cell-apoptosis. Inhibition of apoptosis can result in a number of cancers, autoimmune diseases, inflammatory diseases, and viral infections. It was originally believed that the associated accumulation of cells was due to an increase in cellular proliferation, but it is now known that it is also due to a decrease in cell death. The most common of these diseases is cancer, the disease of excessive cellular proliferation, which is often characterized by an overexpression of IAP family members.
As a result, the malignant cells experience an abnormal response to apoptosis induction: Cycle-regulating genes such as p53, ras or c-myc are mutated or inactivated in diseased cells, and further genes such as bcl-2 also modify their expression in tumors. Some apoptotic factors are vital during mitochondrial respiration e. Apoptosis in HeLa [b] cells is inhibited by proteins produced by the cell; these inhibitory proteins target retinoblastoma tumor-suppressing proteins.
This is an important oncolytic property of CDV: this virus is capable of killing canine lymphoma cells. Oncoproteins E6 and E7 still leave p53 inactive, but they are not able to avoid the activation of caspases induced from the stress of viral infection. These oncolytic properties provided a promising link between CDV and lymphoma apoptosis, which can lead to development of alternative treatment methods for both canine lymphoma and human non-Hodgkin lymphoma.
Defects in the cell cycle are thought to be responsible for the resistance to chemotherapy or radiation by certain tumor cells, so a virus that can induce apoptosis despite defects in the cell cycle is useful for cancer treatment. The main method of treatment for potential death from signaling-related diseases involves either increasing or decreasing the susceptibility of apoptosis in diseased cells, depending on whether the disease is caused by either the inhibition of or excess apoptosis.
For instance, treatments aim to restore apoptosis to treat diseases with deficient cell death, and to increase the apoptotic threshold to treat diseases involved with excessive cell death.
The addition of agents such as Herceptin, Iressa, or Gleevec works to stop cells from cycling and causes apoptosis activation by blocking growth and survival signaling further upstream. Finally, adding p MDM2 complexes displaces p53 and activates the p53 pathway, leading to cell cycle arrest and apoptosis. Many different methods can be used either to stimulate or to inhibit apoptosis in various places along the death signaling pathway.
Apoptosis is a multi-step, multi-pathway cell-death programme that is inherent in every cell of the body. In cancer, the apoptosis cell-division ratio is altered. Cancer treatment by chemotherapy and irradiation kills target cells primarily by inducing apoptosis. On the other hand, loss of control of cell death resulting in excess apoptosis can lead to neurodegenerative diseases, hematologic diseases, and tissue damage. Moreover, there is an inverse epidemiological comorbidity between neurodegenerative diseases and cancer. At the molecular level, hyperactive apoptosis can be caused by defects in signaling pathways that regulate the Bcl-2 family proteins.
Increased expression of apoptotic proteins such as BIM, or their decreased proteolysis, leads to cell death, and can cause a number of pathologies, depending on the cells where excessive activity of BIM occurs.
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Treatments aiming to inhibit works to block specific caspases. Finally, the Akt protein kinase promotes cell survival through two pathways. Akt phosphorylates and inhibits Bad a Bcl-2 family member , causing Bad to interact with the scaffold, resulting in Bcl dissociation and thus cell survival. One of the mechanisms by which T-helper cells are depleted is apoptosis, which results from a series of biochemical pathways: . Cells may also die as direct consequences of viral infections.
Researchers from Kumamoto University in Japan have developed a new method to eradicate HIV in viral reservoir cells, named "Lock-in and apoptosis.
Programmed Cell Death: Methods and Protocols
By suppressing viral budding, the researchers were able to trap the HIV virus in the cell and allow for the cell to undergo apoptosis natural cell death. Associate Professor Mikako Fujita has stated that the approach is not yet available to HIV patients because the research team has to conduct further research on combining the drug therapy that currently exists with this "Lock-in and apoptosis" approach to lead to complete recovery from HIV.
Viral induction of apoptosis occurs when one or several cells of a living organism are infected with a virus , leading to cell death. Cell death in organisms is necessary for the normal development of cells and the cell cycle maturation. Canine distemper virus CDV is known to cause apoptosis in central nervous system and lymphoid tissue of infected dogs in vivo and in vitro. HeLa cell apoptosis caused by CDV follows a different mechanism than that in vero cell lines.
Tools for Studying Cell Death - Enzo Life Sciences
The executioner protein is instead activated by the internal stimuli caused by viral infection not a caspase cascade. The study of apoptosis brought on by Bunyaviridae was initiated in , when it was observed that apoptosis was induced by the La Crosse virus into the kidney cells of baby hamsters and into the brains of baby mice.
OROV is a disease that is transmitted between humans by the biting midge Culicoides paraensis. The Oropouche virus also causes disruption in cultured cells — cells that are cultivated in distinct and specific conditions. An example of this can be seen in HeLa cells , whereby the cells begin to degenerate shortly after they are infected. This type of interaction shows that apoptosis is activated via an intrinsic pathway.
In order for apoptosis to occur within OROV, viral uncoating, viral internalization, along with the replication of cells is necessary. Apoptosis in some viruses is activated by extracellular stimuli. However, studies have demonstrated that the OROV infection causes apoptosis to be activated through intracellular stimuli and involves the mitochondria. The protocols are mostly described in the context of mammalian systems, but also cover other systems such as plants, Drosophila , and yeast.
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Programmed Cell Death: Methods and Protocols is comprised of 20 chapters: Chapters describe apoptosis detection techniques; Chapter describe methods for studying apoptosis associated with various pathologies in different organs including the lymphoid compartment, intestinal epithelium, granulocytes, and cardiomyocytes; Chapter cover protocols and techniques for studying apoptosis in non-mammalian systems; Chapters cover biochemical and biophysical methods for studying Bcl-2 family protein dynamics and protein-protein interactions during apoptosis; and the last four chapters explore protocols that are useful not only in apoptosis research but in wider areas of biological research, such as genome editing, inducible transgenes, and proteomics.
Written in the highly successful Methods in Molecular Biology aeries format, chapters include introductions to their respective topics, lists of the necessary material and reagents, step-by-step, readily reproducible laboratory protocol, and tips on troubleshooting and avoiding known pitfalls. Thorough and cutting-edge, Programmed Cell Death: Methods and Protocols is a comprehensive and valuable resource for researchers, ranging from beginner to expert, in their studies on programmed cell death.
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