Introduction

How do cells regulate and oversee their own mortality? Research on Programmed Cell Death (PCD) and particularly apoptosis has ignited curiosity within the realm of cell biology and the possible ways these intricate processes can make their way into medicine through manipulation. Programmed Cell Death is a vital process, stimulated by pathological and physiological factors, a necessity for limiting the uncontrolled proliferation and replication of cells that could possibly lead to cancer. It inhibits and controls the amount to which cells grow and is a key process in tissue regulation. Studies made in C. elegans (species of nematode used in biological research) have recognized certain genes that itemized which cells must live or die, caspase activation and the final dismantling and removal of these cells.  Regulatory cascades and other regulatory networks are sufficient in the activation and expression of death inducing gene egl-1. This is followed by a protein cascade resulting in the activation of CED-3 (cell death protease). This activates the disintegration of those cells, overlooked by apoptotic factors.

Perhaps the most widely researched type of PCD is Apoptosis. Apoptosis is a self-destructive pathway of cell death, stimulated by cellular injury including external and internal damages to the cell. The complex process of apoptosis involves DNA fragmentation, preceded by condensation of the nucleus and cytoplasm (cell partitioning). During investigations of PCD by means of C. elegans development, from about 1090 somatic cells generated, 131 certainly underwent apoptosis, dying at particular checkpoints. This is a morphologically distinct process and comprises the genetically determined eradication of cells in order to maintain the growth of cells and tissues. Apoptosis can be initiated as a defense mechanism however, there are many triggers that can lead to apoptotic death including radiation and certain drugs. Moreover, some mechanisms of apoptosis, such as ligand binding and protein cross-linking are activated as a consequence of cell receptor expression, particularly Fas or TNF receptors. Overall, apoptosis is a process requiring energy, consisting of the activation of cysteine proteases known as ‘caspases’ followed by a ‘cascade’ that methodically eliminates the targeted cell.  

Non-Immunogenic Death Pathway

Apoptosis is a non-immunogenic death pathway, meaning it does not initiate any immune response, involving the shrinkage and meticulous packing of the target cell. This is proceeded by ingestion and disintegration by phagocytic cells and aids the removal of damaged and infected cells without the release of inflammatory signals.

Apoptosis Mechanism

Activation Mechanisms

·         Intrinsic Pathway: The intrinsic signaling pathway is triggered by internal stress, including damage to DNA, oxidative stress or the withdrawal of certain mitochondrial factors. This pathway in particular is non-receptor induced and involves intracellular signals that have two possible outcomes. The first one includes the activation of apoptosis through the absence of hormones, growth factors and cytokines. (Negative signals). On the other hand, the positive signal is a consequence of factors such as virus, radiation, free radicals and toxins. These stimuli cause a change in the mitochondrial membrane and the initiation of ‘MOMP’(Mitochondrial Outer Membrane Permeabilization). This is the central event in the apoptotic pathway, aided by loss of mitochondrial trans-membrane potential that enables the release of two pro-apoptotic proteins: cytochrome c and the serine protease. Cytochrome c binds to the apoptotic protease activating factor1 or APAF-1 to form the apoptosome. Following the activation of procaspase-9, hetero/homodimers with APAF-1 are produced which in turn activates thecaspase-9. Finally, the caspase-9 activates the effector caspases.   After the death decision, some pro-apoptotic proteins (SMAC/Diablo) cause DNA fragmentation and condensation of chromatin.

·         Extrinsic Pathway: This pathway is triggered by external signals. It is receptor-mediated due to the binding of ligands to their complementary ‘death receptors’. The TNF receptor gene (tumor necrosis factor) gives rise to multiple death receptors, which in their domain, comprise of around 80 amino acids proclaimed the ‘death domain’. This death domain aids in the transfer of signals from the cellular surface to the intracellular signaling pathway. Moreover, the ligands and death receptors usually involved are the FasL /FasR and TNF-a/TNFR1. The binding of the Fas ligand to the receptor leads to the binding of adapter protein FADD. FADD structuring recruits procaspase-8 by dimerization of the death domain which further recruits the inactive caspase-8 (c-FLIP) leading to the formation of death-inducing signaling complex (DISC). Caspase-8 and c-FLIP compete for the adapter protein FADD following the activation of death receptors (through the ligands of the TNF-R family). Consequentially, the extrinsic apoptotic signaling pathway is inhibited.

Morphological Changes

·      During the early stages of apoptosis, cell shrinkage occurs. This involves the cytoplasm becoming denser due to the tight packing of organelles. One of the major characteristic features of apoptosis morphology is pyknosis. Pyknosis is the result of chromatin condensation and aggregation of nuclear material under the nuclear membrane. Some examinations using hematoxylin or eosin stains show a round or oval mass comprising dense purple nuclear chromatin fragments.

·      Blebbing is another morphological change. Blebs are spherical blister-like protrusions on the cell surface. They form as a consequence of cytoskeleton detachment from the plasma membrane. High intracellular pressure as well as actin cortex deterioration initiates the process of blebbing. The contractile activity of myosin filaments is amplified and cortex ruptures. The cytoplasmic flow is driven by hydrostatic pressure as a result of the many processes during apoptosis stages.  

·      Karyorrhexis (Nuclear Fragmentation) is the destructive fragmentation of the nucleus of a dying cell which is proceeded by chromatin distribution irregularly through the cytoplasm. The rupture of the nuclear membrane divides the chromatin into small, basophilic granules.

Phagocytosis of Apoptotic Bodies

·       Apoptotic bodies and cells that are obsolete undergo phagocytosis at the hands of macrophages, neoplastic cells and parenchymal cells. Professional phagocyte such as macrophages and immature DC (imDC) are highly phagocytic. DC are able to phagocytose some antigens as well as apoptotic cells, though they are less efficient than macrophages.

·       Cells are able to display signals, for instance the platelet-endothelial cell adhesion molecule-1 or simply CD31 which activates the detachment of phagocytes and inhibition of the uptake of apoptotic cells. (negative regulation of phagocytosis, maintained by Fgr kinase or SIRP-a receptor interaction prevents ingestion of non-apoptotic cells). During apoptosis, this detachment signal is disabled which sustains the eradication of dying cells, facilitated by CD31.Phosphatidylserine (PtdSer, PS) and annexin-I serve as recognition signals after they translocate on the surface of the exterior membrane.

Regulation

·      Apoptosis is tightly regulated by various proteins, including members of the Bcl-2 protein family. This protein lays the framework for the sustenance of lymphoma cancer cells. This family of proteinshas only 25 members, most of which are known as BH-3 only proteins. They are agents of activation of apoptosis and oversee any cell processes fordysfunction. Along with that, they control the mechanisms of pro-apoptotic proteins for instance Bax or Bak, as well as a considerate amount of anti-apoptotic proteins (e.g Bcl-XL). This process is designated for function on mitochondrial membranes.

·      The second family of proteins involved is the cascade proteolytic enzymes. They aid the execution of apoptosis following confirmation of the death decision. Enzymes responsible for DNA degradation and disassembling of cytoskeleton irreversibly are activated. These components are then packed to prevent their release within tissues.

Conclusion

By manipulating apoptotic processes, we can address certain medical problems. Through careful surveillance of the activity of cells, whether it be amplification or inhibition, we can contribute immensely to disease and medicine.

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Written by: Iman Zahra