PROGRAMMED CELL DEATH (APOPTOSIS)

(Obaidullah khan, Faisalabad)

Author: Obaidullah Khan and Mam Hafiza Masooma, Department of Plant Breeding and Genetics, University of Agriculture Faisalabad.

Apoptosis is the process of programmed cell death that may occur in multicellular organisms. It was first discovered by scientists over 100 years ago. A German scientist Carl Vogt was first to describe the principle of apoptosis in 1842. In Greek, apoptosis translates to "dropping off" of petals or leaves from plants or trees. Cormack, professor of Greek language, reintroduced the term for medical use as it had a medical meaning for the Greeks over two thousand years before. Hippocrates used the term to mean "the falling off of the bones". Galen extended its meaning to "the dropping of the scabs".

Programmed cell death involves a series of biochemical events leading to a characteristic cell morphology and death; in more specific terms, a series of biochemical events that lead to a variety of morphological changes, including blebbing, changes to the cell membrane such as loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. In contrast to necrosis, which is a form of traumatic cell death that results from acute cellular injury, apoptosis, in general, confers advantages during an organism's life cycle. For example, the differentiation of fingers and toes in a developing human embryo occurs because cells between the fingers apoptose; the result is that the digits are separate. Between 50 and 70 billion cells die each day due to apoptosis in the average human adult. For an average child between the ages of 8 and 14, approximately 20 billion to 30 billion cells die a day. In a year, this amounts to the proliferation and subsequent destruction of a mass of cells equal to an individual's body weight.

PROCESS OF APOPTOSIS
The process of apoptosis is controlled by a diverse range of cell signals, which may originate either extracellularly (extrinsic inducers) or intracellularly (intrinsic inducers). Extracellular signals may include toxins, hormones, growth factors, nitric oxide orcytokines, and therefore must either cross the plasma membrane or transduce to effect a response. These signals may positively (i.e., trigger) or negatively (i.e., repress, inhibit, or dampen) affect apoptosis. A cell initiates intracellular apoptotic signalling in response to a stress, which may bring about cell suicide. The binding of nuclear receptors by glucocorticoids, heat, radiation, nutrient deprivation, viral infection, hypoxia and increased intracellularcalcium concentration, for example, by damage to the membrane, can all trigger the release of intracellular apoptotic signals by a damaged cell. A number of cellular components, such as poly ADP ribose polymerase, may also help regulate apoptosis.Before the actual process of cell death is precipitated by enzymes, apoptotic signals must cause regulatory proteins to initiate the apoptosis pathway. This step allows apoptotic signals to cause cell death, or the process to be stopped, should the cell no longer need to die. Several proteins are involved, but two main methods of regulation have been identified: targeting mitochondria functionality, or directly transducing the signal via adaptor proteins to the apoptotic mechanisms. Another extrinsic pathway for initiation identified in several toxin studies is an increase in calcium concentration within a cell caused by drug activity, which also can cause apoptosis via a calcium binding protease calpain.

Many pathways and signals lead to apoptosis, but there is only one mechanism that actually causes the death of a cell. After a cell receives stimulus, it undergoes organized degradation of cellular organelles by activatedproteolytic caspases. A cell undergoing apoptosis shows a characteristic morphology:
1. Cell shrinkage and rounding are shown because of the breakdown of the proteinaceous cytoskeleton by caspases.
2. The cytoplasm appears dense, and the organelles appear tightly packed.
3. Chromatin undergoes condensation into compact patches against the nuclear envelope in a process known as pyknosis, a hallmark of apoptosis.
4. The nuclear envelope becomes discontinuous and the DNA inside it is fragmented in a process referred to as karyorrhexis. The nucleus breaks into several discrete chromatin bodies or nucleosomal units due to the degradation of DNA.
5. The cell membrane shows irregular buds known as blebs.
6. The cell breaks apart into several vesicles called apoptotic bodies, which are then phagocytosed.
Cell Termination

Apoptosis occurs when a cell is damaged beyond repair, infected with a virus, or undergoing stressful conditions such as starvation. Damage to DNA from ionizing radiation or toxic chemicals can also induce apoptosis via the actions of the tumor-suppressing gene p53. The "decision" for apoptosis can come from the cell itself, from the surrounding tissue, or from a cell that is part of the immune system. In these cases apoptosis functions to remove the damaged cell, preventing it from sapping further nutrients from the organism, or halting further spread of viral infection.Apoptosis also plays a role in preventing cancer. If a cell is unable to undergo apoptosis because of mutation or biochemical inhibition, it continues to divide and develop into a tumor. For example, infection by papillomaviruses causes a viral gene to interfere with the cell's p53protein, an important member of the apoptotic pathway. This interference in the apoptotic capability of the cell plays a role in the development of cervical cancer.

Homeostasis
In the adult organism, the number of cells is kept relatively constant through cell death and division. Cells must be replaced when they malfunction or become diseased, but proliferation must be offset by cell death. This control mechanism is part of the homeostas is required by living organisms to maintain their internal states within certain limits. Some scientists have suggested homeodynamics as a more accurate term. The related term allostasis reflects a balance of a more complex nature by the body. Homeostasis is achieved when the rate of mitosis (cell division resulting in cell multiplication) in the tissue is balanced by the rate of cell death. If this equilibrium is disturbed, one of two potentially fatal disorders occurs:
 the cells divide faster than they die, resulting in the development of a tumor.
 the cells divide slower than they die, causing cell loss.
Homeostasis involves a complex series of reactions, an ongoing process inside an organism that calls for different types of cell signaling. Any impairment can cause a disease. For example, dysregulation of signaling pathway has been implicated in several forms of cancer. The pathway, which conveys an anti-apoptotic signal, has been found to be activated in pancreatic adenocarcinoma tissues.

Development
Programmed cell death is an integral part of both plant and animal tissue development. Development of an organ or tissue is often preceded by the extensive division and differentiation of a particular cell, the resultant mass is then "pruned" into the correct form by apoptosis. Unlikenecrosis, cellular death caused by injury, apoptosis results in cell shrinkage and fragmentation. Such shrinkage and fragmentation allow the cells to be phagocytosed and their components reused without releasing potentially harmful intracellular substances such as hydrolytic enzymes into the surrounding tissue.During development, apoptosis is tightly regulated and different tissues use different signals for inducing apoptosis. In birds, bone morphogenetic proteins (BMP) signaling is used to induce apoptosis in the interdigital tissue. In Drosophila flies, steroid hormonesregulate cell death. Developmental cues can also induce apoptosis, such as the sex-specific cell death of hermaphrodite specific neuronsin C. elegans males through low TRA-1 transcription factor activity.

Defective Apoptotic Pathways
The many different types of apoptotic pathways contain a multitude of different biochemical components, many of them not yet understood. (Thompson et al., 1995) . As a pathway is more or less sequential in nature, it is a victim of causality; removing or modifying one component leads to an effect in another. In a living organism this can have disastrous effects, often in the form of disease or disorder. A discussion of every disease caused by modification of the various apoptotic pathways would be impractical, but the concept overlying each one is the same: the normal functioning of the pathway has been disrupted in such a way as to impair the ability of the cell to undergo normal apoptosis. This results in a cell that lives past its "use-by-date" and is able to replicate and pass on any faulty machinery to its progeny, increasing the likelihood of the cell becoming cancerous or diseased.A recently-described example of this concept in action can be seen in the development of a lung cancer called NCI-H460. (Yang et al., 2003). The X-linked inhibitor of apoptosis protein (XIAP) isoverexpressed in cells of the H460 cell line. XIAPs bind to the processed form of caspase-9, and suppress the activity of apoptotic activator cytochrome c, therefore overexpression leads to a decrease in the amount of pro-apoptotic agonists. As a consequence, the balance of anti-apoptotic and pro-apoptotic effectors is upset in favour of the former, and the damaged cells continue to replicate despite being directed to die.

HIV Progression
The progression of the human immunodeficiency virus infection to AIDS is primarily due to the depletion of CD4+ T-helper lymphocytes, which leads to a compromised immune system. One of the mechanisms by which T-helper cells are depleted is apoptosis, which results from a series of biochemical pathways (Judie et al., 2003).
1. HIV enzymes deactivate anti-apoptotic Bcl-2 This does not directly cause cell death, but primes the cell for apoptosis should the appropriate signal be received. In parallel, these enzymes activate pro-apoptotic procaspase-8, which does directly activate the mitochondrial events of apoptosis.
2. HIV may increase the level of cellular proteins which prompt Fas-mediated apoptosis.
3. HIV proteins decrease the amount of CD4 glycoprotein marker present on the cell membrane.
4. Released viral particles and proteins present in extracellular fluid are able to induce apoptosis in nearby "bystander" T helper cells.
5. HIV decreases the production of molecules involved in marking the cell for apoptosis, giving the virus time to replicate and continue releasing apoptotic agents and virions into the surrounding tissue.
6. The infected CD4+ cell may also receive the death signal from a cytotoxic T cell.
Cells may also die as a direct consequence of viral infection.
Viral Infection
Viruses can trigger apoptosis of infected cells via a range of mechanisms including:
 Receptor binding.
 Activation of protein kinase R (PKR).
 Interaction with p53.
 Expression of viral proteins coupled to MHC proteins on the surface of the infected cell, allowing recognition by cells of the immune system (such as Natural Killer and cytotoxic T cells) that then induce the infected cell to undergo apoptosis. (Everett et al., 1999).
Most viruses encode proteins that can inhibit apoptosis. Several viruses encode viral homologs of Bcl-2. These homologs can inhibit pro-apoptotic proteins such as BAX and BAK, which are essential for the activation of apoptosis. Examples of viral Bcl-2 proteins include the Epstein-Barr virus BHRF1 protein and the adenovirus E1B 19K protein. Some viruses express caspase inhibitors that inhibit caspase activity and an example is the CrmA protein of cowpox viruses. Whilst a number of viruses can block the effects of TNF and Fas. For example the M-T2 protein of myxoma viruses can bind TNF preventing it from binding the TNF receptor and inducing a response. Furthermore, many viruses express p53 inhibitors that can bind p53 and inhibit its transcriptional transactivation activity. Consequently p53 cannot induce apoptosis since it cannot induce the expression of pro-apoptotic proteins. The adenovirus E1B-55K protein and the hepatitis B virus HBx protein are examples of viral proteins that can perform such a function. ( Wang et al., 1995).
Interestingly, viruses can remain intact from apoptosis particularly in the latter stages of infection. They can be exported in the apoptotic bodies that pinch off from the surface of the dying cell and the fact that they are engulfed by phagocytes prevents the initiation of a host response. This favours the spread of the virus. (Hay et al., 2002).
References:
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2. Yang L, Mashima T, Sato S, et al. (February 2003). "Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide". Cancer Res. 63 (4): 831–7. PMID 12591734.
3. Takaoka A, et al. (2003). "Integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence". Nature 424 (6948): 516–23. doi:10.1038/nature01850. PMID 12872134.
4. Judie B. Alimonti, T. Blake Ball, Keith R. Fowke (2003). "Mechanisms of CD4+ T lymphocyte cell death in human immunodeficiency virus infection and AIDS". J Gen Virology (84): 1649–61. doi:10.1099/vir.0.19110-0. PMID 12810858.
5. Everett, H. and McFadden, G. (1999). "Apoptosis: an innate immune response to virus infection". Trends Microbiol 7 (4): 160–5. doi:10.1016/S0966-842X(99)01487-0. PMID 10217831.

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