Apoptosis, also called programmed cell death, is a normal process where cells are induced to die in order to maintain an appropriate cell population during development and aging. It also serves as an immune defense when cells are under attack by pathogens, noxious agents or diseases. At certain conditions, cells are marked with apoptosis markers and the induced to go under apoptotic pathways which eventually results in self-destruction without affecting other adjacent cells. Apoptosis and necrosis, a process in which the cells are forced to die under physical or chemical pressures, are easy to be mistaken from each other since both of them induces cell deaths. Both of the processes can occur independently, sequentially or even simultaneously.
The key difference between them is the way the cells die. Induced by apoptosis cell die by cell shrinkage, therefore it does not release its cytoplasmic content to the surrounding environment and later will be phagocytosed by macrophages or the adjacent cells without causing inflammation reactions. In the opposite, cell induced by necrosis die by bursting out its cytoplasmic content, which can lead to domino effect of inflammation to the whole tissue or organ. The specific morphologies of apoptotic cell are cell shrinkage, membrane bledding, chromosome condensation, nuclear fragmentation and DNA laddering. Apoptosis is considered as an irreversible process with caspase activation leading to cell suicide.
There are three pathways involved in cell apoptosis, including extrinsic or death receptor pathway, intrinsic or mitochondrial pathway and perforin/granzyme pathway. These three pathways are stimulated by different stimuli and have several distinguish proteins involved. However, they eventually lead to the activation of a common protein kinase called caspase-3, which initiates the executive apoptotic pathway which leads to cell death. The stimuli of the extrinsic pathway are death ligands, such as Fas. When bound to its receptor, Fas can activate the adaptor protein called FADD, meanwhile, the binding of TNF ligand to its receptor activates that TRADD protein.
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This protein then recruits the binding of FADD and RIP proteins, then the complex comes to have an association with procaspase 8 forming death-inducing signaling complex (DISC) which in turn phosphorylates the caspase-3 protein. The perforin/granzyme pathway is stimulated by viral infections or tumors and induced under cytotoxic T cell signaling. When stimulated, T cell exerts its cytotoxic effects on targeted cells via pathway involving the secretion of transmembrane pore-forming molecule called perforin together with cytoplasmic granules such as granzyme A or granzyme B. These proteins can act as endonuclease that cleave proteins into fragments which can later activate the caspase-3 protein. There are a wide range of stimuli that can activate apoptotic intrinsic pathway, including radiation, hypoxia, viral infection or free radical.
These stimulations causes the opening of mitochondria permeability transition pore results in the loss of membrane potential which lead to the leaking of the lumen content, including cytochrome c. Cytochrome c is well known for its function in cellular respiration inside the lumen of mitochondria, however, when released into the cytoplasm, it forms complex with Apaf-1 and procaspase 9 to form apoptosome which will later trigger the cascade activations of caspase 9 and caspase-3. As mention above, all three apoptosis pathways lead to the activation of caspase-3, which can phosphorylate CAD endonuclease that cleaves proteins such as cytokeratin or PARP. These proteins can cause cellular morphological and biochemical changes, eventually lead to cell death.
Currently in scientific research, there are three assays heavily used for the detection of apoptotic markers by FCM, including Annexin/7-AAD (BD PharmingenTM PE Annexin V Apoptosis Detection Kit I, BD Biosciences, San Jose, CA), caspase-3 expression (CaspGLOWTM Fluorecein Active-Caspase-3 Staining Kit, Introvigen) and measurement of reactive oxygen species (ROS) (DCFDA-based Cellular ROS Detection Kit, Abcam, Cambridge, MA). One of the biochemical feature of early apoptosis is the externalization of phosphatidylserine, a component of plasma membrane normally inward-facing, to be expressed on the outer layers of plasma membrane. This phospholipid therefore acts as a ligand for the phagocytosis signaling of adjacent cells to digest the phosphatidylserine expressing cells.
Because of its feature, phosphatidylserine is immensely used as the marker for apoptosis marker. Annexin V, a recombinant protein that strongly and specifically binds to phosphatidylserine, together with 7-ADD which is a fluorescent DNA binding dye indicating the membrane impermeability, can be used to identify the apoptotic cells at different states. Cells that stained positive for Annexin V and negative for 7-AAD is considered at early apoptosis. Cells that stained both positive for Annexin V and 7-AAD are either at the end stage of apoptosis or already death.
Cells that stained both negative for the reagents are alive and not undergoing measurable apoptosis. Another method to track down the apoptotic cells is measuring the activated caspase-3 by using fluorescent conjugated antibody against it. This can be achieved by performing intracellular FCM staining. The last assay used in apoptosis identification is ROS detection. ROS is a short-lived and highly reactive molecule that play essential roles in cell development such as differentiation, proliferation, cell signaling and immune defense. However, when there is an imbalance in ROS generation and antioxidant to neutralize it, ROS can cause serious cellular damages, including protein, DNA, lipid and organelles. Since ROS is a type of free radical, therefore its excessive level can trigger the activation of intrinsic apoptosis pathway.
