Apotosis

Apoptosis, or programmed cell death, is a process fundamental to the normal development and homeostasis of multicellular organisms. Deregulation of programmed cell death leads to a number of human diseases, including cancer, neurodegenerative disorders, and acquired immunodeficiency syndrome. The cell death machinery comprises effectors, activators, and negative regulators.   

Apotosis should be distinguished from the more random process of necrotic cell death, which is closely associated with the inflammatory responses that are commonly observed during an infectious diesease. Necrosis elicits inflammation, whereas the recognition of apoptotic cells leads not only to phagocytosis but also to downregulation of inflammation via the anti-inflammatory cytokines TGF-β and IL-10 due to interaction of the phosphatidylserine (PS) phagocyte receptor with apoptotic cells.

See also     Apoptosis Protocols    Apoptosis Assays   

Apoptosis is necessary in Human Development and Maintenance

Apoptosis is vital in the following processes:

• T lympocytes development: Most immature T cells are useless (incorrect rearrangement of the T cell receptor) or potentially detrimental (self-reactive) to the organisms. More than 95% of thymocytes that immigrate into the thymus are eliminated by positive and negative selection during their development.

• Killing of target cells by CTLs and NK cells:

• Immune privilege: Cellular immune response reactions and their associated inflammatory responses can cause nonspecific damage to nearby tissues. Although most organs can tolerate such inflammation, some, such as the eye and testis, cannot. These organs have mechanisms to protect themselves against unwanted immune reactions. Although inflammatory cells can enter these organs, they are killed by FasL expressed in the organs. This suggests a use for FasL as an immunosuppressive agent to target activated effector cells in transplantation. Interestingly, several groups have found that some tumor cells become resistant to Fas-induced apoptosis and constitutively express FasL. FasL expressed on tumor cells then counterattacks CTL and NK cells by binding Fas on their surfaces to cause apoptosis. This mechanism may account for the ability of tumor cells to evade immune destruction.

• Infectious Disease: It is clear that apoptosis has a direct role in many infectious diseases, especially those caused by viruses, intracellular protozoans and intracellular bacteria.

(1) Inducing Signals

Multiple physiologic and pathologic signals are capable of inducing apoptosis in disparate cell types, and multiple signal transduction processes result in apoptosis in different tissues. The process of apoptosis can be triggered by pleiotropic ways, including the following:

• physical agents such as X-ray, y-irradiation

• chemical agents such as cellular toxins, hormones, and natural signaling molecules such as tumor necrosis factor (TNF), transforming growth factor Β (TGF-B), and Fas ligand (FasL, CD95).

• Growth factor withdrawl such as nerve growth factor withdrawal from neurons

(2) Signaling Pathways in Apoptosis

Pathways for apoptosis induction or inhibition originate from both extrinsic and intrinsic signalling systems. Extrinsic signalling pathways are initiated by ligand binding to cell-surface receptors (e.g., Fas ligand binding to Fas/Apo1/CD95). Intrinsic signalling involves the activation and oligomerization of proteins (e.g., BAX) that are capable of binding and destablizing mitochondrial membranes. In most cases, both extrinsic and intrinsic apoptosis require the activation of a family of proteases (CASPASES) and culminate in the participation of nucleases and other destructive enzymes that eliminate cells without eliciting inflammation. Both apoptotic pathways converge on the activation of caspase-3 which is the executioner of apoptosis.

• extrinsic pathway: The extrnsic pathway is mediated by ligation of a death receptor upon its binding to a death-inducing ligand that results in the activation of caspase-8, which activates caspase-3. Extrinsic initiation of apoptosis occurs by stimulation of sets of surface receptors (e.g., CD95 (fas/APO-1 or TNFR) by their cognate ligands. This results in trimerization and docking of proteins containing so-called death domains which in turn activate large amounts of caspase 8, an initiator caspase, followed directly by cleavage of downstream effector caspases in Type I cells. By contrast, Type II cells produce little activate caspase 8 and therefore relay on a mitochondrial amplification step.

The extrinsic pathway can be induced by interactions between cytokines  (such as TNF-alpha, interferon-gamma and TGF-B) and death receptors, resulting in activation of Casp-8. Click here for Cytokine Induction of Apoptosis:

• intrinsic pathway: The instrinsic pathway is mediated by agents such as stress stimuli and toxins that cause the release of cytochrome c from mitochondria into the cytoplasms and result in activation of caspase-9 which activates caspase-3. The Intrinsic induction of apoptotis starts within the cell and can occur by direct activation of caspases or a variety of stress-related mechanisms. The intrinsic pathway is triggered in response to a variety of apoptotic stimuli including anticancer agents, oxidative damage, UV irradiation and growth factor withdrawal and is mediated through mitochondria. These stimuli induce the loss of mitochondrial membrane integrity and result in the critical event -- release of multiple molecules, including cytochrome c (cyt c), which associates with Apaf-1 and Casp-9 to promote caspase activation.

The release of cytochrome c is largely controlled by members of proteins of the Bcl-2 family, which can act to promote or to inhibit this release. On a molecular level, free cytosolic cytochrome c initiates the formation of a signaling complex (called the apoptosome) that encompasses the molecules Apaf-1 and the proteases caspase-9 and caspase-3. In the formation of this complex, caspase-9 is activated with in turn activates caspase-3. Active caspase-3 then cleaves cellular substrates to bring about the morphological changes of apoptosis such as nuclear condensation.

• granzyme B pathway, where the cytotoxic cell protease granzyme B is delivered to sensitive target cells.

Although apoptosis through death receptors does not appear to require the involvement of mitochondria, several levels of cross-talking exist between these two pathways. For example, the Bcl-2 family plays an important role in this cross-talking by acting on mitochondria. Active capase-8, the downstream effector caspase of the extrinsic pathway can cleave the cytosolic Bid into the BH3-only domain-containing, proapoptotic, truncated tBid fragment which can translocate into mitochondria, where it binds to its mitochondrial proapoptotic partner Bax or Bak to trigger the release of cyt c into the cytosol. In certain cell lines, overexpression of Bcl-2/Bcl-xl prevents the efflux of cyt c from the mitochondria, and can prevent cell death receptor-induced apoptosis.

(3) Effector or Execution Pathways in Apoptosis

The signals which induce apoptosis and the signalling traduction processes appear to be transduced to common effector or execution pathways. For example, each of the pathways above converges to a common execution phase of apoptosis that requires activation of caspases 3 and 7 from their inactive zymogen form to their processed, activate form. In apoptosis, caspases function in both initiation and execution of cell disassembly in response to apoptotic signal.  

The activation of the cell death pathway depends on both the triggering stimulus and the cell type, and in many forms of apoptosis cytochrome c release from mitochondria is important for activation of downstream caspases. Programmed death of cells is affected by many of the same gene products controlling cell cycle progression.

Despite the large role which caspases play in apoptosis, noncapse proteases such as cathepsins D and B, calpains and various serine proteases have been reported as essential downstream effectors of caspases, for instance, in TNF-mediated apoptosis. Apoptosis can even occur in the complete absence of caspase activation.

Cathepsin D: has been proposed to mediate a regulated type of programmed cell death, initiated by various cytokines.

The known signaling pathways induced by various apoptotic stimuli converge into a common death pathway either at a mitochondrial step or finally at a step at which Asp-Glu-Val-Asp (DEVD)-specific caspase-3-like cysteine proteases are activated. Active caspase 3 like proteases cleave a limited set of cellular proteins, and the resulting inactivation/activation of substrates leads to the typical apoptotic morphology of the dying cell. Although the activation of a caspase cascade has been considered a hallmark of apoptosis, apoptotic pathways not requiring known caspases have been reported. For example, a proapoptotic protein Bax, which targets mitochondrail membranes; a topoisomerase I poison, camptothesin; and nitric oxide have been reported to induce apoptosis-like cell death with caspase activation.

Topoisomerase poisons particularly topoisomerase I inhibitors, induce apoptosis in many cells that lack functional p53. Topoisomerases are ubiquitous enzymes that modulate the topographic structure of DNA by transiently introducing breaks in the DNA backbone.       

Regulation of Apoptosis

Use of Apoptosis in the Treatment of Human Disease

Not surprisingly, agents that induce apoptosis in cancer cells have attracted a great deal of attention. This makes sense, because if you can induce cancer cells to undergo programmed cell death, you can potentially eliminate the cancer.

Morphological Changes Associated with Apoptosis

In apoptosis a cell shrinks and condenses. The cytoskeleton collapses, the nuclear envelope disassembles and the nuclear DNA breaks up into fragments. The cell surface also displays a phosphatidylserine (PS) that cause the dying cell to be rapidly phagocytosed. All of these changes are very different from what happens in cell necrosis which is where a cell wells and bursts due to acute injury.

Markers of Apoptosis

• Cleavage of poly-(ADP-rebose) polymerase has been used as a surrogate marker for the entry of cells into the irreversible execution phase of apoptosis, even though this event appears likely to be an epiphenomenon

• cleavage of the nuclear matrix protein lamin B is thought to be related to nuclear chromatin changes that occur during apoptosis.