Apoptosis is a type of typical genetically designed cell death in which an aging cell shrinks and its residual pieces are phagocytosed without any inflammatory response.
Kerr, Wyllie, and Currie first used the word apoptosis to characterize a morphologically unique form of cell death in an article published in 1972. It comprises of a sequence of biochemical changes that result in alterations in the morphology or demise of the cell. In a typical mature human being, it results in the death of 50 to 70 billion cells per day. Cells experience a highly controlled process for the planned elimination of cells from the body, which is also known as cellular suicide.
Table of Contents
Why do cells undergo apoptosis?
- As part of the cell cycle, most cells have a death process built in.
- This process enables the body to eliminate superfluous or infected cells.
- Apoptosis is thought to be an important component of many processes, including regular cell cycle, immune system growth and function, embryonic development, and chemical-induced cell death.
- Apoptosis is a component of development because it is required for the differentiation of a bulk of tissue into distinct groups.
- Apoptosis happens in cells that have been contaminated with viruses or are malignant. When a cell recognizes a defect in its DNA and is unable to fix it, this process occurs.
- Apoptosis is also an important component of the immune system because it clears pathogen-specific immune cells after the foreign substance has been cleared from the body.
- This also aids in the removal of immune cells that may respond against bodily cells and create autoimmune illnesses.
- Another reason for apoptosis is to keep bodily balance by removing old cells to create room for new ones.
Apoptosis mechanisms
The process of apoptosis is highly complex and sophisticated, involving an energy-dependent series of molecular events.
Apoptosis is accomplished through three distinct paths that utilize various methods. All three of these routes eventually meet at the same final pathway, resulting in the successive degradation of cellular organelles.
1. Extrinsic or death receptor pathway
- Transmembrane receptor-mediated interactions are involved in the extrinsic route that starts apoptosis.
- These interactions occur between molecules and their associated death receptors, which are all members of the tumor necrosis factor (TNF) family.
- The "death domain" is a cysteine-rich cytoplasmic region with about 80 amino acids that is shared by all members of the TNF receptor family.
- The death domain is critical in relaying the death signal from the cell membrane to the intracellular signaling networks.
- The processes or interactions that occur during the extrinsic phase of apoptosis involve two models: FasL/FasR and TNF-/TNFR1 models, both of which include receptor aggregation and binding.
- When a ligand binds to a receptor, cytoplasmic adaptor proteins are triggered, causing the receptors to display death domains.
- The binding of FasL to FasR activates the adapter protein FADD, whereas the binding of TNF ligand (TNF) to TNF receptor (TNFR1) activates the adaptor protein TRADD, which in turn activates FADD and RIP.
- These events induce the death effector domain to dimerize, prompting FADD to bind to procaspase-8.
- The association results in the formation of a death-inducing signaling complex (DISC), which results in the auto-catalytic stimulation of procaspase-8.
- When caspase-8 is initiated, apoptosis enters the final phase, also known as the execution phase.
2. The intrinsic or mitochondrial pathway
- The intrinsic route that starts apoptosis consists of a succession of non-receptor-mediated processes that generate intracellular signals and operate directly on cell targets.
- This pathway includes processes started by the mitochondria.
- The variables that start the intrinsic route generate intracellular signals that can operate positively or negatively.
- Negative signals include the lack of certain growth factors, cytokines, and hormones, which can result in the failure of death programs to inhibit, causing apoptosis. In layman's terms, factor withdrawal results in a lack of apoptotic suppression and following stimulation of apoptosis.
- Radiation, poisons, hypoxia, hyperthermia, virus diseases, and free radicals are examples of favorable variables.
- All of these variables induce alterations in the inner mitochondrial membrane, resulting in the opening of the mitochondrial permeability transition (MPT) pore and the release of two major groups of pro-apoptotic proteins into the cytosol from the intermembranous region.
- The first group comprises of cytochrome c, which binds and activates Apoptotic protease-activating factor - 1 (Apaf-1) and procaspase-9, creating an apoptosome protein complex.
- The apoptosome cleaves procaspase into caspase 9, which then cleaves and triggers procaspase to create effector caspase 3.
- Other proteins in the first category include SMACs (second mitochondria-derived activator of caspases) and HtrA2/Omi, which support apoptosis by inhibiting the action of IAPs. (inhibitors of apoptosis proteins).
- The second set of pro-apoptotic proteins is released from the mitochondria during apoptosis, but this happens after the cell has decided to die.
- These proteins translocate to the nucleus, where they induce DNA breakage and nuclear chromatin condensation.
3. Perforin/granzyme pathway
- The perforin/granzyme pathway is a new route used by cytotoxic T lymphocytes to kill tumor cells and virus-infected cells.
- This includes the secretion of perforin, a transmembrane hole-forming molecule, followed by the discharge of cytoplasmic granules through the opening and towards the target cell.
- Granules contain two critical serine proteases, granzyme A and granzyme B, which trigger various proteins in the process.
- Granzyme B cleaves proteins at aspartate residues, activating procaspase-10 and cleaving factors such as ICAD (Inhibitor of Caspase Activated DNAse).
- Granzyme B has also been shown to use the mitochondrial route to amplify the death signal by inducing cytochrome c release.
- Granzyme B, on the other hand, can immediately trigger caspase-3. The execution step of apoptosis is directly induced in this mechanism.
- Granzyme A is also required for cytotoxic T-cell mediated apoptosis and triggers caspase-independent pathways.
- As granzyme A enters the cell, it initiates DNA nicking by the DNAse enzyme, which stops cancer by inducing tumor cell apoptosis.
- Granzyme A protease cleaves the SET complex, which suppresses DNAse enzyme synthesis.
- The SET complex proteins work together to preserve chromatin and DNA integrity. Thus, granzyme A inactivation of the SET complex adds to apoptosis by interfering with the preservation of DNA and chromatin structure integrity.
4. Execution pathway
- Both the extrinsic and intrinsic routes terminate at the execution phase, which is regarded as the final pathway of apoptosis.
- The activation of different caspases, which trigger cytoplasmic endonucleases and proteases, initiates this period of apoptosis.
- Nuclear material is degraded by cytoplasmic endonucleases, whereas nuclear and cytoskeletal proteins are degraded by proteases.
- Caspase-3 is the most significant executioner caspase protein, and it is triggered by any of the initiator caspases. (caspase-8, caspase-9, or caspase-10).
- Caspase-3 exactly triggers the Caspase-activated DNase endonuclease. (CAD). By destroying chromosomal DNA within the nuclei, CAD induces chromatin condensation.
- Caspase-3 also triggers cytoskeletal rearrangement and the cell's breakdown into apoptotic bodies.
- Gelsolin, an actin-binding protein, is one of activated caspase-3's essential substrates. Caspase-3 cleaves gelsolin, and the cleaved pieces of gelsolin cleave actin strands, causing cytoskeleton disruption and the creation of apoptotic bodies.
- Phosphatidylserine appears on the exterior leaflet of apoptotic cells in the final phases of apoptosis.
- Noninflammatory phagocytic identification is facilitated, enabling for early absorption and disposal.
- No inflammatory reaction is triggered because the process occurs without the release of cellular components.
Inhibition of apoptosis
- Apoptosis inhibition suppresses cell death signaling pathways, allowing malignant cells to avoid apoptosis.
- Different classes of proteins, known as anti-apoptotic factors, serve as negative regulators of apoptosis. These proteins include IAPs and Bcl-2.
- IAP (Inhibitor of apoptosis) proteins are a class of proteins that act as negative controllers of caspases and cell death.
- In humans, the IAP group comprises of eight proteins, each of which has a distinct BIR (Baculovirus IAP Repeat) domain that attaches to caspases and other apoptosis-related proteins.
- XIAP and other proteins attach caspase-9 and caspase-3, inhibiting their activation and stopping apoptosis.
- Bcl-2, another protein that regulates mitochondrial membrane permeability, can be either pro- or anti-apoptotic.
- Anti-apoptotic proteins include Bcl-2, Bcl-x, and BAG, which suppress cytochrome c release and also alter the permeability of the mitochondrial membrane, thereby preventing the intrinsic route of apoptosis.
- Cancers such as leukemia and multiple myeloma are primarily caused by cells' capacity to resist death.
- Inhibiting apoptosis also causes the defense system to lose function. A mutation in the inhibitor protein XIAP causes an uncommon genetically driven immunodeficiency.
Regulation of apoptosis
- Apoptosis is controlled by a number of proteins and genes. Specific protein families are involved in the control of apoptosis at different stages.
- IAPs and Bcl-2 are two of the most essential proteins involved in determining whether apoptosis will be completed or inhibited.
- A protein called c-FLIP inhibits the extrinsic route of apoptosis by binding to FADD and caspase-8, making them inactive.
- Another extrinsic pathway apoptosis control method includes a protein called Toso, which inhibits Fas-induced apoptosis in T cells by inhibiting caspase-8 activation.
- Members of the Bcl-2 family play an essential part in the intrinsic pathway's regulation and management.
- The Bcl-2 protein family regulates mitochondrial membrane permeability, and the proteins can be either pro- or anti-apoptotic.
- The Bcl-2 family of proteins regulates apoptosis by controlling the release of cytochrome c from the mitochondria via changes in mitochondrial membrane permeability.
- Proteins such as Puma and Noxa are pro-apoptotic factors that promote apoptosis by blocking the action of anti-apoptotic factors.
- Smac, a group of mitochondrial proteins that induce apoptosis by inhibiting the activity of IAPs in the mitochondrial pathway.
Apoptosis assays
- Because the apoptosis process is closely controlled at various stages, the activity of various proteins involved can be assessed.
- Because the processes of apoptosis and necrosis may intersect, it is critical to validate the method of cell death using two distinct tests.
- The first article indicates the onset of apoptosis, while the second identifies the execution or terminal phase.
- Apoptosis tests are classified into six categories, which are as follows:
1. Cytomorphological altercation
- Light microscopy enables the visualization of apoptotic cells in hematoxylin and eosin-stained tissue slices.
- This technique identifies cells in the later stages of apoptosis, but not cells in the early stages of apoptosis.
- The gold standard for confirming apoptosis is transmission electron microscopy (TEM).
- Apoptosis cells show several structural features in TEM. Among these personalities are:
- electron-rich nucleus (marginalization of the nucleus in the early phase)
- Nuclear fragmentation, even late in the cell breakdown phase,
- undamaged cell membrane,
- disordered cytoplasmic organelles,
- huge transparent vacuoles, and
- phosphatidylserine at the cell surface.
- As apoptosis progresses, these cells lose cell-to-cell adhesions and split from adjacent cells.
- The cell will eventually split into apoptotic bodies with complete cell membranes and cytoplasmic structures with or without nuclear pieces.
2. DNA fragmentation
- Another way of detecting apoptosis is the DNA laddering approach, which visualizes the products of endonuclease cleavage during apoptosis.
- This entails separating DNA from a disintegrated cell lysate using agarose gel electrophoresis.
- The resulting DNA bands create a DNA ladder, which can be used to identify apoptosis in tissues with a large number of apoptotic cells.
- However, because DNA fragmentation happens only in the later phases of apoptosis, this method cannot identify cells in the early stages.
- TUNEL (Terminal dUTP Nick End-marking) is another technique that identifies endonuclease cleavage products by enzymatically marking the ends of DNA strands.
- To bind dUTP to the 3′ end of the DNA segments, terminal transferase is used.
- The dUTP is then tagged with various probes so that it can be detected using light microscope, fluorescence imaging, or flow cytometry.
- Although this method is quick and can be completed in a few hours, it has the potential to produce false-positive findings from necrotic cells.
3. Detection of caspases, cleaved substrate, regulators and inhibitors
- Caspase activity assays of various kinds are available to identify more than 13 known caspases implicated in apoptosis.
- Some immunoassays can identify cleavage substrates like PARP as well as well-known cell changes like phosphorylated histones.
- Caspase activation can be detected using a number of techniques such as western blot, immunoprecipitation, and immunohistochemistry.
- Apoptosis PCR microarray is a relatively novel technique that employs real-time PCR to detect the expression of approximately 112 apoptosis-related genes.
- The microarrays are intended to generate an expression map of genes encoding important receptors, ligands, intracellular regulators, and transcription factors involved in the control of programmed cell death.
- This methodology can also be used to evaluate the genes implicated in anti-apoptosis.
- This method can only provide an approximation of the amount of apoptotic cells and must thus be used in conjunction with other tests.
4. Membrane altercations
- An-nexin V can identify the presence of phosphatidylserine residues on the exterior plasma membrane of apoptotic cells in tissues, fetuses, or cultured cells.
- The apoptotic cells are first coupled with FITC-labeled Annexin V before being observed under fluorescent microscope.
- This method has a drawback in that the membranes of dead cells may also be tagged. To prevent this, necrotic cells can be marked with membrane-impermeant nucleic acid dyes such as propidium iodide and trypan blue to identify membrane integrity loss.
- The lack of these pigments, on the other hand, can identify the membrane integrity of apoptotic cells.
- The migration of phosphatidylserine to the cell membrane will also enable the unidirectional transfer of some dyes into the cell.
- As a result, the cell may collect dye and decrease in bulk. As a consequence, the cell dye substance becomes more condensed and can be seen under a microscope.
5. Detection of Apoptosis in Whole Mounts
- To view whole preparations of embryos or tissues, dyes such as acridine orange (AO), Nile blue sulfate (NBS), and neutral red (NR) can be used.
- These acidophilic dyes prefer to cluster in regions with strong lysosomal and phagocytotic activity.
- This method should be used in conjunction with another test because it cannot distinguish between apoptotic and microorganism debris.
- However, these colors have some drawbacks, such as acridine orange being mutagenic and toxic, and NBS and neutral red not penetrating deep into the tissues and being lost during processing.
- Lyso-Tracker Red, another dye, can be used in conjunction with laser confocal photography to provide 3-dimensional imagery of apoptotic cells.
6. Mitochondrial assays
- Mitochondrial cytochrome c release assays allow for the identification of changes during the early phases of the intrinsic pathway.
- Laser scanning confocal microscopy (LSCM) produces narrow optical slices of living cells, which are then used to track different mitochondrial processes in intact single cells over time.
- This method detects mitochondrial permeability, inner mitochondrial membrane depolarization, mitochondrial redox state, Ca2+fluxes, and reactive oxygen species.
- However, because these alterations occur during necrosis, they cannot be used solely to identify apoptosis.
- Other mitochondrial colors are also available to detect the redox potential or metabolic activity of mitochondria in cells. However, a caspase detection assay should be used in conjunction with this method to identify the cause of apoptosis.
- Fluorescence and electron imaging can also be used to measure cytochrome c emission from the mitochondria of moving or fixed cells.
- Fluorescence and confocal imaging can also identify pro-apoptotic or anti-apoptotic regulator proteins such as Bax, Bid, and Bcl-2. However, because fluorescent protein tags may change protein interactions, they should be supported by other assays for proof.
Apoptosis significance/ Applications/ Roles
- Apoptosis is a process in which many cells die as a result of planned cell death, adding to the creation of different tissues and organs from a single clump of tissue. It can even be used as a predictor of prenatal defects.
- The frequent elimination of old cells from the body allows the body to create new cells, thereby assisting in the maintenance of the cell population in the body. Depending on the sort of cells, the inability to do so could have dramatic repercussions.
- Apoptosis aids in the elimination of obsolete and injured cells from the body. Simultaneously, virus-infected cells and those that cannot be fixed are eliminated via apoptosis.
- The immune system frequently uses apoptosis to determine whether freshly created cells are self-destructive or not. If immune cells are discovered to be harmful to the body's cells, they are removed, avoiding the development of autoimmune illnesses.
- Apoptosis is an important process in the body's defense system. Various cytotoxic cells, such as T lymphocytes, are created in preparation during foreign substance infection. Once the invader has been taken from the body, the remaining pathogen-specific immune cells are eliminated through apoptosis.
- Apoptosis has also been linked to the regulation of thymocyte growth, T cell formation, and the integration of different immune reactions.
- Apoptosis is also involved in the elimination of approximately half of the neurons generated during early embryonic development, as well as the creation of reproductive organs.
- Excess apoptosis can lead to neurodegenerative illnesses, whereas inadequate apoptosis can lead to cancer and inflammatory diseases.
Examples of Apoptosis
Metamorphosis of the adult frog from tadpole
- In frogs, apoptosis occurs when many structures are killed and reabsorbed as the tadpole frog metamorphoses into a mature form.
- Tadpoles have gills, a tail, and even appendages that are taken from the body as they develop into adult frogs.
- All of these components are known to be destroyed by various apoptosis processes.
The nervous system in humans
- A significant number of cells (nearly 50%) are eliminated by apoptosis during the early growth of the nervous system in the human embryo.
- The precise cause for the loss of so many neurons is unknown. However, it has been suggested that because neurons make complicated connections, a greater number of cells are made to guarantee the process's efficacy.
- As a consequence, a greater number of neurons are created, which are subsequently eliminated to sustain the amount of neurons in the nervous system.
Sloughing off of endometrium
- Apoptosis is the process by which layers of cells in the uterine mucosa are removed.
- Menstruation, an essential event in the female reproductive system, is caused by the periodic loss of cells in the endometrium and corpus luteum.
Formation of hands and feet
- Organs such as hands and feet originate as a flat pile of tissue during the embryonic growth of a multicellular creature.
- As development proceeds, the tissue separates into individual digits and toes, with apoptosis removing the cells that link them.
- This is an illustration of apoptosis occurring during the development and shaping of various organs from a single clump of tissue.
Apoptosis and Cancer
- Cancer is thought to be the product of several genetic changes in a normal cell that cause it to turn into a malignant one, with the avoidance of cell death being an important change during the process.
- To put it another way, a normal cell must survive planned cell death in order to be changed into a malignant cell.
- Apoptosis is typically initiated by DNA damage that cannot be repaired by any cellular process.
- However, if the DNA damage causes harm to the genes responsible for apoptosis, the process of apoptosis may not occur.
- Similarly, a mismatch in pro-apoptotic and anti-apoptotic proteins in the cell may prevent cell death. Resistance to chemotherapy has been found in pancreas cancer cells with abnormal IAP family expression.
- Another possibility is that cells are evading apoptosis due to decreased caspase activity or cell death signaling. One example is caspase-9 downregulation in individuals with stage II colon cancer.
- Because apoptosis is thought to be one of the causes of cancer in many cells, drugs or therapy methods that return apoptotic signaling pathways to normalcy may have the potential to eliminate cancer cells.
- As a result, a number of treatment methods targeting anti-apoptotic proteins and other apoptosis-inhibiting factors have been discovered.
Apoptosis in Plants
- The method and cause of apoptosis in plants are comparable to those of programmed cell death in animals.
- However, some variations exist because plants have a cell membrane and lack an immune system that uses apoptosis to remove different particles.
- When discussing this process in plants, the favored term is programmed cell death.
- Plants' planned cell demise is regulated by cellular oxidative state, phytohormones, and DNA methylation.
- The mechanism of this process varies from that of animals in that protease protein is used instead of caspases, which produce morphological alterations in the plant cell in animals.
- The stimulation of an enzyme causes structural changes in the cell, causing it to break down and integrate into a vacuole.
- As the cell expires from planned cell death, the center vacuole ruptures.
- The morphological changes associated with planned cell death in plants include cytoplasm compaction and vacuolization in apoptotic cells, as well as the emergence of distinct single-membrane compartments holding active organelles in the vacuoles.
- Nuclear DNA synthesis halts, chromatin consolidation and buildup in the nucleus, nuclear DNA fragmentation in the nucleolus, and extensive synthesis of mitochondrial DNA in vacuolar vesicles.
Apoptosis vs Necrosis (17 Differences)
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