The term apoptosis was first introduced in an article in 1972 by Kerr, Wyllie, and Currie to describe a morphologically distinct type of cell death. It consists of a series of biochemical changes that lead to changes in the morphology or death of the cell. It results in the death of 50 to 70 billion cells per day in an average adult human being. Also called “cell suicide” since cells undergo a highly regulated process for the programmed removal of cells from the body.
Why do cells undergo apoptosis?
- Most cells have an underlying apoptosis mechanism as part of the cell cycle.
- This mechanism allows the body to get rid of unnecessary cells or infected cells.
- Apoptosis is considered an important part of many processes, including the normal cell cycle, proper development and function of the immune system, embryonic development, and chemical-induced cell death.
- Apoptosis is part of development, as it is necessary in differentiating the mass of tissue into different groups.
- Apoptosis occurs in cells that could have been infected with the virus or even become cancerous. This process typically occurs when the cell detects defects in DNA and cannot repair it.
- Apoptosis is also an essential part of the immune system, as it kills pathogen-specific immune cells when foreign particles are removed from the body.
- It also helps in killing immune cells which can react against cells in the body and can cause autoimmune diseases.
- Another reason for apoptosis is to maintain homeostasis in the body by removing old cells to make room for new ones.
The process of apoptosis is highly complex and sophisticated, involving an energy-dependent chain of molecular events.
Three different pathways operate on different mechanisms to achieve apoptosis. All three pathways converge in the same terminal pathway, resulting in the gradual degradation of cellular organisms.
Extrinsic or death receptor pathway
- The external pathway that initiates apoptosis involves transmembrane receptor-mediated interactions.
- These interactions occur between ligands and their associated death receptors that are all parts of the tumor necrosis factor (TNF) family.
- All members of the TNF receptor family share a common cysteine-enriched extracellular domain with approximately 80 amino acids, known as the “death domain”.
- The death signal plays an important role in transmitting from the cell surface to intracellular signaling pathways.
- Events or interactions occurring in the outer phase of apoptosis involve two models; FasL / FasR and TNF-α / TNFR1 models, including clustering and binding of receptors and their ligands.
- Upon ligand binding, cytoplasmic adapter proteins are activated, which causes the receptors to display a death domain.
- The binding of FasL to FasR leads to activation of the adapter protein FADD whereas binding of TNF ligand (TNF α) to TNF receptor (TNFR1) results in activation of FADD and RIP in the binding of adapter protein TRDD.
- These events cause dimerization of the defector domain, thereby binding FADD to procaspase-8.
- As a result of the binding, a death-inducing signaling complex (DISC) is formed, resulting in auto-catalytic activation of procaspase-8.
- Once caspase-8 is activated, the terminal phase or execution phase of apoptosis is triggered.
The intrinsic or mitochondrial pathway
- The intrinsic pathway that initiates apoptosis involves a series of non-receptor mediated processes that produce intracellular signals and act directly on targets within the cell.
- This pathway includes events initiated by mitochondria.
- The initiating factors of the intrinsic pathway produce intracellular signals that can act positively or negatively.
- Negative signs include the absence of certain growth factors, cytokines, and hormones that can lead to the failure of death inhibition programs, which can lead to apoptosis. In simple terms, removal of factors leads to loss of apoptotic suppression and subsequent activation of apoptosis.
- Positively acting factors include radiation, toxins, hypoxia, hyperthermia, viral infections, free radicals, etc.
- All of these factors cause changes in the internal mitochondrial membrane that cause the opening of the mitochondrial permeability transition pore (MPT) and release two main groups of pro-apoptotic proteins from the intermembrane space in the cytosol.
- The first group consists of cytochrome c that binds to and activates the apoptotic protease activation factor, forming the apoptotic protease activation factor-1 (Apaf-1), as well as a protein complex terminated apoptosome.
- Apoptosomes form the crespase in the active form, caspase 9, which further activates cleavage and is declared in Prospector caspase 3.
- The first group also contains other proteins such as SMAC (the second mitochondria-derived caspase catalyst) and HtrA2 / Omi that promote apoptosis by inhibiting the activity of IAPs (apoptosis protein inhibitors).
- The second group of pro-apoptotic proteins are released from the mitochondria during apoptosis but do so as part of the terminal phase after the cell dies.
- These proteins become nuclei and cause DNA fragmentation and condensation of the peripheral nuclear chromatin.
Perforin / Granzyme route
- The Perforin / Granzyme pathway is a novel pathway employed by cytotoxic T lymphocytes that enhances their cytotoxic effects on tumor cells and virus-infected cells.
- It contains the secretion of the transmembrane pore-forming molecule, perforin, with subsequent release of cytoplasmic granules through the pore and toward the target cell.
- The granules contain two important serine proteases; Granzyme A and Granzyme B which activate different proteins in the pathway.
- Granzyme B binds to proteins on aspartate residues and therefore activates procaspase-10 and can cleave factors such as ICAD (active inhibitors of caspase).
- It has also been observed that granzyme B may use the mitochondrial pathway for amplification of the death signal by induction of cytochrome c release.
- But granzyme B can also directly activate caspase-3. In this pathway, there is a direct induction of the execution phase of apoptosis.
- Granzyme A has an essential role in cytotoxic T cell-induced apoptosis and activates the caspase-independent pathway.
- As granzyme A approaches the cell, it activates DNA nicking by the DNA enzyme that inhibits cancer through tumor cell adoption.
- The granzyme cleaves a protease SET complex that inhibits the production of DNA enzymes.
- Proteins that make the SET complex together protect chromatin and DNA structure. Thus, inactivation of the set complex by granzyme A contributes to apoptosis by inhibiting the maintenance of DNA and chromatin integrity.
- Both external and internal pathways end at the point of the execution phase, which is considered the terminal pathway of apoptosis.
- This phase of apoptosis begins with the activation of various caspases that activate cytoplasmic endonuclease and protease.
- Cytoplasmic endonucleases degrade nuclear material, whereas proteases degrade nuclear and cytoskeletal proteins.
- Caspase-3 is the most important protein of hangman caspases and is activated by any initiator caspases (caspase-8, caspase-9, or caspase-10).
- Caspase-3 accurately activates the endonuclease Caspase-activated DNase (CAD). CAD then causes chromatin condensation by degrading chromosomal DNA within the nucleus.
- Caspase-3 also causes cytoplasm reorganization and cell disintegration.
- The actin-binding protein, gelsolin, is considered one of the important substances of active caspase-3. The caspase-3 cleaves gelsolin and the cleaved fragment of gelsolin, in turn, cleaves actin filaments, resulting in disruption of the cytoskeleton and formation of an apoptotic body.
- In the later stages of apoptosis, there is the presence of phosphatidylserine on the outer leaflets of apoptotic cells.
- This facilitates noninflammatory phagocytic recognition, allowing their early regeneration and disposal.
- As the process occurs without the release of cellular components, no inflammatory response is detected.
Inhibition of apoptosis
- Inhibition of apoptosis inhibits the cell death signaling pathway, which helps tumor cells avoid apoptosis.
- Different groups of proteins act as negative regulators of apoptosis that have been classified as anti-apoptotic factors such as IAPs and Bcl-2.
- The IAP (inhibitors of apoptosis) proteins represent a group of negative regulators of both caspases and cell death.
- The IAP group in humans contains 8 proteins, all of which have a characteristic BIR (baculovirus IAP repeat) domain that binds with caspases and other proteins involved in apoptosis.
- Proteins such as XIAP bind caspase-9 and caspase-3, thus inhibiting their activation and inhibiting apoptosis.
- Another factor, Bcl-2, regulates mitochondrial membrane permeability and can be either pro-apoptotic or anti-apoptotic.
- Anti-apoptotic proteins include certain proteins such as Bcl-2, Bcl-x, and BAG that inhibit the release of cytochrome c and also modulate the permeability of the mitochondrial membrane, thus disrupting the intrinsic pathway of apoptosis.
- The ability of cells to survive apoptosis is the leading cause of cancer such as leukemia and multiple myeloma.
- Inhibition of apoptosis also induces a loss of immune function by the immune system. Mutation of the inhibitory protein XIAP leads to a rare genetically mediated immunity.
Regulation of apoptosis
- Many proteins and genes regulate apoptosis. Specific families of proteins are involved in the regulation of apoptosis at various stages.
- Of all the factors, IAP and Bcl-2 are the most important proteins that determine whether apoptosis is about to be completed or inhibited.
- The external pathway of apoptosis is disrupted by proteins called c-FLIP that will bind to FADD and caspase-8, making them ineffective.
- Another mechanism of apoptosis regulation in the extrinsic pathway involves a protein called TSO, which inhibits Fas-induced apoptosis in T cells by inhibition of caspase-8 activation.
- In the internal pathway, members of the Bcl-2 family play an important role in the regulation and control of the pathway.
- The Bcl-2 family of proteins regulates mitochondrial membrane permeability, and the protein can be either pro-apoptotic or anti-apoptotic.
- Bcl-2 family proteins regulate apoptosis by regulating the release of cytochrome c from mitochondria through alteration of mitochondrial membrane permeability.
- Proteins such as Puma and Noxa are members of pro-apoptotic factors that facilitate the activation of apoptosis by inhibiting the action of anti-apoptotic factors.
- Smac, a group of proteins originating from mitochondria, promotes apoptosis by inhibiting the action of IAPs in the mitochondrial pathway.
- Because the process of apoptosis is tightly controlled at various points, it is possible to evaluate the activity of the various proteins involved.
- It is necessary to confirm the mechanism of cell death by two different assays as apoptosis and necrosis overlap.
- The first essay traces the early events of apoptosis, while the second identifies the execution or terminal stage.
- Apoptosis assays are divided into six different groups, which are:
- The observation of hematoxylin and eosin-stained tissue sections with light microscopy allows visualization of apoptotic cells.
- This method detects cells in subsequent events of apoptosis, but cells are not recognized in the early stage of apoptosis.
- Transmission electron microscopy (TEM) is the gold standard for confirmation of apoptosis.
- In TEM, several structural features are detected in cells undergoing apoptosis. These characters include:
- Electron-dense nucleus (the nucleus of the nucleus in the early phase)
- nuclear fission
- Intact cell membrane becomes too late in the phase of cell dissolution
- Disorganized cytoplasmic organelle
- Big clear space
- Phosphatidylserine on the cell surface.
- With the progression of apoptosis, these cells will lose cell-to-cell adhesion and dissociate from neighboring cells.
- Eventually, the cell will fragment into apoptotic bodies with intact cell membranes and include cytoplasmic organelles with or without nuclear fragments.
- The DNA laddering technique is another way to detect apoptosis that visualizes the products of endonuclease cleavage during transplanting.
- This involves the extraction of DNA from a lysed cell homogenate separation by agarose gel electrophoresis.
- The resulting bands of DNA from a DNA ladder that can be used to detect apoptosis in tissues where the number of apoptotic cells is high.
- However, DNA fragmentation occurs only during the later stages of apoptosis, and thus it cannot detect cells at an early stage.
- Another method is the TUNEL (terminal dUTP nick end-labeling) method that detects endonuclease cleavage products by labeling enzymes at the ends of DNA strands.
- A terminal transfer is used to attach dUTP to the 3 of-end of DNA segments.
- The DUTP is then examined in a variety of ways to allow detection by light microscopy, fluorescence microscopy, or flow cytometry.
- Although this technique is fast and can be conducted in a few hours, it can give false-positive results from necrotic cells.
Detection of caspases, cleaved substrates, regulators and inhibitors
- A variety of caspase activity assay is available that detect more than 13 known caspases involved in apoptosis.
- Some immunoassays can detect cleaved substrates such as PARP and known cell modifications such as phosphorylated histones.
- A variety of assays, including Western blot, immunoprevention, and immunohistochemistry, can be used to detect caspase activation.
- Apoptosis PCR microarray is a comparatively new method that uses real-time PCR to indicate the expression of approximately 112 genes involved in apoptosis.
- Microarrays are designed to produce expression profiles of genes that encode essential receptors, ligands, intracellular regulators, and transcription factors involved in the regulation of programmed cell death.
- Genes involved in anti-apoptosis can also be assessed with this method.
- This technique can only provide an estimate of the number of apoptotic cells and as such should be the case with other assays.
- The presence of phosphatidylserine residues on the outer plasma membrane of apoptotic cells can be detected through N-nexin V in tissues, embryos, or cultured cells.
- Apoptotic cells are first bound to FITC-labeled annexin V and then visualized with fluorescent microscopy.
- This technique has one disadvantage because the membranes of necrotic cells can also be labeled. To avoid this, necrotic cells can be stained with membrane-amplified nucleic acid dyes such as propidium iodide and trypan blue to detect loss of membrane integrity.
- Conversely, the membrane integrity of apoptotic cells can be detected by the absence of these dyes.
- The movement of phosphatidylserine outside the cell membrane will also lead to the transport of certain dyes in the cell in a unidirectional manner.
- Thus, the cell can accumulate dye and shrink in volume. As a result, the cell dye material becomes more concentrated and can be visualized even with light microscopy.
Detection of Apoptosis in Whole Mounts
- Dyes such as Eridine Orange (AO), Nile Blue Sulfate (NBS), and Neutral Red (NR) can be used to visualize the entire volume of embryos or tissues.
- Since these dyes are acidophilic, they concentrate in areas of high lysosomal and phagocytotic activity.
- This technique should be combined with another assay because it cannot separate apoptotic debris from the debris of microorganisms.
- However, there are some disadvantages to these dyes, where acridine orange is mutagenic and toxic, and NBS and neutral red do not penetrate deeply into tissues and may be lost during preparation.
- Another dye, the lyso-tracker red, can be used with laser confocal microscopy to provide 3-dimensional imaging of apoptotic cells.
- Mitochondrial assays demonstrating cytochrome c release allow the detection of changes during the early stages of the intrinsic pathway.
- Laser scanning confocal microscopy (LSCM) creates thin optical slices of living cells that are then used to monitor different mitochondrial events in monolithic single cells at the same time.
- This technique measures parameters such as mitochondrial permeability, depolarization of the inner mitochondrial membrane, mitochondrial redox status, Ca 2+ flux, and reactive oxygen species.
- However, these changes also occur during necrosis and thus cannot be specifically used as apoptosis detection.
- Other mitochondrial dyes that measure the redox potential or metabolic activity of mitochondria in cells are also available. To determine the mechanism of apoptosis, however, a caspase detection assay must be performed with this technique.
- Cytochrome c release from mitochondria of living or fixed cells can also be accepted using fluorescence and electron microscopy.
- Pro-apoptotic or anti-apoptotic regulator proteins such as Bax, Bid, and Bcl-2 can also be detected using fluorescence and confocal microscopy. However, fluorescent protein tags can alter interactions between proteins and thus have to be accompanied by other assays for confirmation.
- Apoptosis is essential during development where multiple cells progressively undergo cell death, thus contributing to the formation of different tissues and organs from the same mass of tissue. It can also be used as an important determinant of fetal abnormalities.
- Regular removal of old cells from the body enables the body to produce new cells to help maintain the cell population in the body. The inability to do so can have dramatic consequences depending on the type of cells.
- Apoptosis helps in removing unnecessary and damaged cells from the body. At the same time, cells that are infected with a virus, and which cannot be repaired, are removed through apoptosis.
- Apoptosis is often used by the body’s immune system to test whether newly formed cells are self-destructive. If immune cells are found to be destructive against the body’s cell, they are removed, preventing the possibility of autoimmune diseases.
- Apoptosis plays an important role in the body’s immune system. Various cytotoxic cells, such as T lymphocytes, are produced in advance during infection by foreign material. Once the invader is removed from the body, existing pathogen-specific immune cells are removed from the body through apoptosis.
- Apoptosis has also been observed in regulating thymocyte development and the size of T cells and the coordination of various immune responses.
- Apoptosis is also involved in the removal of approximately 50% of neurons during early embryonic development and in the formation of reproductive parts.
- Additional apoptosis can result in neurodegenerative diseases, and conditions such as cancer and autoimmune diseases can result in decreased apoptosis.
Examples of apoptosis
Metamorphosis of the adult frog from tadpole
- Apoptosis is seen in frogs where many structures are destroyed and reabsorbed while the tadpole frog transforms into an adult form.
- The tadpoles have gills, tails, and even wings, which are removed from the body while transforming into an adult frog.
- All of these structures are known to be destroyed by various mechanisms of apoptosis.
The nervous system in humans
- During the early development of the nervous system in the human embryo, a large number of (about 50%) cells are removed through apoptosis.
- The exact reason behind the death of this many neurons is not fully known. However, it has hypothesized that because neurons make complex connections, large numbers of cells are produced to ensure the efficiency of the process.
- As a result, a large number of neurons will be produced, which are subsequently removed to maintain the number of neurons in the nervous system.
Sloughing off of endometrium
- The process of removing layers of cells in the endometrium of the uterus is by apoptosis.
- Periodic loss of cells in the endometrium and corpus luteum forms the basis for menstruation, an important process in the female reproductive system.
Formation of arms and legs
- During embryonic development of a multicellular organism, organs such as hands and feet begin as a flat mass of tissue.
- As development progresses, the tissue separates into individual fingers and toes, where the cells connecting them are removed by apoptosis.
- This is an example of apoptosis involved in the formation and size of various organs from a singular mass of tissue.
Apoptosis and Cancer
- Cancer is thought to be the result of several genetic changes in a normal cell, transforming it into an incurable, which necessitates cell death as a necessary change during the process.
- In simple words, to convert a normal cell into a malignant cell, it must avoid programmed cell death.
- Apoptosis is usually triggered by DNA damage that usually cannot be repaired by any mechanism in the cell.
- However, if DNA damage results in damage to the genes responsible for apoptosis, then the process of apoptosis cannot occur.
- Similarly, imbalances in pro-apoptotic and anti-apoptotic proteins in the cell can also prevent apoptosis of cells. Abnormal expression of the IAP family in pancreatic cancer cells has been observed due to resistance to chemotherapy.
- Another reason may be reduced caspase function or reduced cell death signaling, which may also lead to evasion of apoptosis by cells. An example of this is the decline of caspase-9 in patients with stage II colorectal cancer.
- Because apoptosis is considered one of the reasons for the involvement of cancer in many cells, drugs, or treatment strategies, it may have the ability to eliminate cancer cells to restore the apoptotic signaling pathway to normality.
- Thus several treatment strategies have been developed targeting anti-apoptotic proteins and other factors that prevent apoptosis.
Apoptosis in plants
- The process and mechanism of apoptosis in plants have some similarities with the mechanism of cell death programmed in animals.
- However, some differences arise due to the presence of a cell wall in plants and the lack of an immune system that uses apoptosis to remove various particles.
- Programmed cell death is the preferred term when describing this process in plants.
- The process of programmed cell death in plants is regulated by the cellular oxidative state, phytohormones, and DNA methylation.
- The mechanism of this process is the use of caspases (in animals) instead of protease proteins in animals, which cause morphological changes in the plant cell.
- Activation of the enzyme induces various morphological changes in the cell, causing the cell to break down itself and join the vacuole.
- The central vacuole then bursts because cell death occurs at the end of the programmed cell death.
-The morphological changes associated with programmed cell death in plants include:
- Condensation and immunization of cytoplasm in apoptotic cells
- Specific fragmentation of cytoplasm and the presence of unique single-membrane vesicles containing active organelles in the vacuole
- End of nuclear DNA synthesis
- Condensation and accumulation of chromatin in the nucleus
- Fission of nuclear DNA in the nucleus
- Intensive synthesis of mitochondrial DNA in vacuole vesicles.