Apoptosis

Apoptosis is of Greek origin, having the meaning "falling off or dropping off". Apoptosis is an energy dependent process by which individual cells undergo programmed cell death in response to various extrinsic and intrinsic factors without inducing inflammatory responses. Normal development of organ requires not only cell division and cell differentiation but also elimination of cell by apoptosis. The term programmed cell death was introduced in 1964, proposing that cell death during development is not of accidental nature but follows a sequence of controlled steps leading to locally and temporally defined self-destruction.

Morphological Features of Apoptosis

The process of apoptosis can be divided into three stages: Induction Stage In this stage there is initial signal for apoptosis by variety of stimuli like various stresses including deprivation of serum, growth factors or cytokines, heat shock and various carcinogenic reagents. Execution Stage This stage involves the classic morphological and biochemical changes. Morphological changes includes condensation and peripheralization of chromatin, vacuolization and loss of cytoplasm, fragmentation of nucleus, corn paction of organelles, the disestablishment of communication with neighboring cells, fusion of the endoplasmic reticulum with the outer cell membrane. The biochemical change involves the activation of a specific series of cytoplasmic cellular proteases and endonucleases, caspase. The activation of these self- catalytic caspase in the cytoplasm has been identified. Degradation Stage This occurs due to the various morphological and biochemical changes mentioned above. Finally there is fragmentation of the cell DNA and other macromolecules into size of nucleosome units due to the activation of endogenous nuclear endonucleases. Water is extracted from the cell resulting into marked decrease in cell size and increase in density. The increase in density has been used to isolate apoptotic from non-apoptotic cell. The shrunken apoptotic cells subsequently fragmented into sealed vesicles and thus formation of numerous membranes bound apoptotic bodies containing DNA which are engulfed by the surrounding cells. During necrosis, the cellular contents are released uncontrolled into the cell's environment which results in damage of surrounding cells and a strong inflammatory response in the corresponding tissue.

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Detection of Apoptosis
The DNA fragmentation which occurs in apoptosis can be visualized by agar gel electrophoresis. The reduction in cell size and volume in apoptosis can be studied by microscopy of flow cytometry. A number of dyes such as ethidium bromide or propidium iodide can enter in the apoptotic cell due to altered permeability, and thus number of dead cells can be obtained in flow cytometry. The following methods have been developed to quantify DNA fragmentation: F ACS (Fluorescent Activation Cell Sorter) Analysis In this method the isolated nuclei are stained with various dyes. The DNA content of apoptotic nuclei is Terminal Deoxy Nucleotide Transferase (Tdt) Mediated DUTPdigoxigenin Nick and Labeling (TUNEL) Assay This method is based on in situ labeling of DNA fragmentation sites in the nuclei of intact fixed cells. Tissue sections can also be stained by this technique.

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Suppression of Apoptosis
Sometimes through an active programme suppression of apoptosis is needed to maintain the cells in quiescent state. The execution of the quiescence program appears to be essential for the long term survival of peripheral lymphocytes and is dependent upon signals transduced through the Band T cell antigen receptors. In addition, the transcription factor LKLF is required component of this programme in T-cells.

Necrosis
Necrosis means the death of cells or tissues in the living body. Here cell dying by this process has no control over its fate. In some cases death is rapid or sudden, but in others various degenerative changes takes places, such as cloudy swelling and fatty degeneration, before the cell or tissues dies, and the process of dying is gradual one. Here the cell death is due to leakage of the lysosomal enzymes into the cytoplasm, swelling of the cell and eventual rupture of the plasma membrane. When a cell die in the living body there follows short lapse of time during which comparatively little histological change takes place and the cell presents an appearance identical with that shown by the healthy cell which has been killed by fixation. There are several causes, which initiates necrosis. These include followings: Micro-Organism and Their Products Invasion of cells by different micro-organisms, such as Mycobacterium tuberculosis, Corynebacterium diphtheriae and toxins of Clostridium sp. May cause necrosis. Physical Agents Various physical agents like electricity, extreme heat and cold and X-rays and prolonged pressure (by ligature or tumors) may lead to necrosis of the cells. Chemical Agents Various chemicals such as carbolic acids, mineral acids and caustics act directly on cells resulting into necrosis of cells. Enzymes Fat splitting enzymes from the pancreas which gives rise to fat necrosis. Interference with Nutrition Interference with nutrition by arrest of circulation in tissues where a collateral blood supply is absent or insufficient to ensure the life of the tissues. When such obstruction occurs, infarction results and one of the main features of infarction is necrosis.

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Pressure Necrosis It occurs as a result of long continued pressure at a particular place, generally seen in orthopaedic patients, who have to lie down for a longer period of time. In them pressure necrosis may be seen in mild form like bed sores. Lack of Nerve Supply It has been observed that limbs and other parts suffer atrophy and necrosis when deprived of normal innervations.

Difference between Apoptosis and Necrosis

There are followings difference between apoptosis and necrosis

Apoptosis
1. Apoptosis occurs at single cell level. 2. It is an active type of programmed cell death that is highly regulated and involves the activation of cascade of molecular events leading to cell death. 3. It is a programmed cell death and is initiated as a response to external signals from other cells, or as a result of changes in the intracellular macromolecules. 4. Here during cell death there is no leakage of the cell content, instead there is fragmentation of DNA, the cytoplasm shows blabbing and increased granularity and there is fracturing of the cell into small apoptotic bodies containing DNA. 5. Here no inflammatory changes are occurred. 6. in apoptosis there is shrinkage of cells occurred.

Necrosis
1. Necrosis occurs in a group of cells or in tissues at a particular locus. 2. It is a passive degeneration of cells characterized by catastrophic toxic events. 3. Necrosis is caused by external injury, damage or microbial agents. 4. In necrosis cell death is due to leakage of the lysosomal enzymes into the cytoplasm, swelling of the cell and eventual rupture of the plasma membrane. 5. Necrosis is always observed accompanied by inflammatory changes. 6. in necrosis there is swelling of cell occurred.

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The Significance of Apoptosis

During development many cells are produced in excess which eventually undergo programmed cell death. For example: During limb formation separate digits evolve by death of inter digital mesenchymal tissue. Ablation of cells no longer needed such as the amphibian tadpole tail during metamorphosis. Demise of cells allows sculpturing of hollow structures. Formation of reproductive organs e.g. process occurs in ovaries of the female when the number of immature egg cells in the ovaries is gradually reduced from midway in pregnancy until birth. Massive cell death occurs during early development of the nervous system. In the development of the brain during which half of the neurons that is initially created will die in later stages when the adult brain is formed. Cells of an adult organism constantly undergo physiological cell death which must be balanced with proliferation in order to maintain homeostasis in terms of constant cell number. Apoptosis involve in the homeostasis of the immune system. Several millions of B and T cells are generated everyday and the majority of those die during maturation (death by neglect, negative selection or by AICD of peripheral immune cells). Cells with severely damaged DNA that cannot be repaired appropriately usually are removed by apoptosis Inappropriate mitogenic signaling that is in conflict with the environmental or cellular status of the cell usually results in cell cycle arrest or apoptosis Auto reactive cells of the immune system are deleted by apoptosis Elimination of infected cells Defects in apoptosis can result in cancer, autoimmune diseases and spreading of viral infections, while neurodegenerative disorders, AIDS and ischemic diseases are caused or enhanced by excessive apoptosis It helps in adaptation of an organism to environment and resolution of inflammation by safe elimination of unwanted cells It helps in removing the damaged, infected and potentially neoplastic cells and thus protects the human beings and different livestock from various diseases including cancer.

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Signals for the Cell to Undergo Apoptosis

Apoptosis in a cell can be triggered by verity of signals. This include a balance between the withdrawal positive signals i.e. require for continue survival of the cell and the recipient of negative signals. Examples of positive signals are of followings: Growth factors for neurons. IL-2 (Inter Lucent 2) for maturation of lymphocytes. Examples of negative signals are of followings: Increase level of oxidants within a cell. DNA damage by UV rays or any chemotheripic drug.

Genes Regulating the Apoptosis

Apoptosis is a genetically controlled process and is controlled by certain genes. These genes either inhibit or promote the apoptosis. Promoters: BAX Fas TP53 Inhibitors: BCl-2

Pathway of Apoptosis
There are mainly three pathways of apoptosis:

1. Intrinsic Pathway
This pathway also called as the mitochondrial pathway or granzyme pathway. In this pathway the cytosol of T-lymphocytes secretes the granzyme and perforase. The perforase make pores in the mitochondrial membrane and granzyme enter in mitochondria through these pores. Hare the granzyme make the release of cytochrome C. The cytochrome C combines with the apoptosome which is formed by the Apaf-1 and procaspase-1. The apoptosome with cytochrome C act on inactive procaspase-13 and make it activated i.e. caspase-13.

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The activated caspase-13 causes apoptosis.

2. Extrinsic Pathway
In this pathway the inactive procaspase-8 is activated to caspase-8 by FADD. The activated caspase-8 further act on inactive procaspase-3 and activated it to caspase-3 which causes apoptosis.

3. Apoptosis Inducing Factors (AIF)

AIF is a protein that is normally located in the intermembraneous space of mitochondria. When a cell received a signal that it is the time to die AIF is released from the mitochondria. AIF migrates to nucleus and bind to DNA.

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This cause destruction of DNA leads to cell death.

Major Apoptotic Signaling Pathways

Apoptosis can be induced in response to various signals from inside and outside the cell. Signals emanating from death receptors initially activate the Death Inducing Signaling Complex (DISC) which mediates activation of the initiator caspase-8. Activated caspase-8 initiates a caspase cascade by processing the effector caspases-3, -6, and 7 which in turn cleave a number of protein substrates. Cleavage of caspase substrate eventually leads to the characteristic morphological and biochemical features of apoptosis. In some cell systems, this direct caspase cascade is sufficient to elicit apoptosis on its own(type 1 signaling), whereas in other cases the signal coming from the DISC must be amplified by the proteolytic activation of the BH3-only protein Bid by caspase-8 with subsequent induction of apoptotic events at the mitochondria (type 2 signaling). Mitochondrial apoptotic signaling includes the release of cytochrome c from the mitochondrial inter membrane space to the cytosol where it contributes to the formation of the apoptosome which consists of cytochrome c, Apaf-1 and dATP. The apoptosome activates caspase-9 which is another initiator caspase and thus is able to mediate the caspase cascade by activating caspase-3. Another mitochondrial pro apoptotic factor is Smac which acts by inhibiting the IAPs from blocking caspase activity. IAPs are a family of proteins with anti-apoptotic activity by directly inhibiting caspase. IAP expression can be up regulated in response to survival signals such as those coming from growth factor receptors, e.g. by activation of the transcription factorNF-kB, therefore providing a means to suppress apoptosis signaling. Of central importance are the anti-apoptotic Bcl-2 family members such as Bcl-2 and Bcl-XL which counteract the action of BH3-only proteins such as Bid but also of pro apoptotic Bax and Bak and thus can inhibit mitochondrial pro apoptotic events. Apoptotic signals coming from the inside of the cell frequently have their origin within the nucleus, being a consequence of DNA damage induced by irradiation, drugs or other sort of stress. DNA damage in most cases eventually results in the activation of the p53 transcription factor which promotes expression of proapoptotic Bcl-2 members and suppresses anti apoptotic Bcl-2 and Bcl-XL. Other organelles besides mitochondria and the nucleus, such as the ER and lysosomes also have been implicated in apoptotic signaling pathways, and it should be kept in

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mind that presumably hundreds of proteins are part of an extremely fine-tuned regulatory network consisting of pro- and anti-apoptotic factors.

Regulation of Apoptosis by the Bcl-2 Family

In a viable cell, the proapoptotic Bcl-2 family members Bax, Bak, and BH3-only proteins are antagonized by anti-apoptotic members such asBcl-2. In response to an apoptotic stimulus, BH3-only members are activated by transcriptional up regulation (Bax, Noxa, and Puma), sub cellular relocalization (Bim, Bmf), dephosphorylation (Bad), or proteolysis (Bid). Activated BH3-only proteins prevent anti apoptotic Bcl-2 members from inhibiting proapoptotic members. In addition, they might directly induce a conformational change of Bax and Bak which subsequently oligomerize and insert into the mitochondrial membrane where they form pores either by themselves or by associating with the permeability transition pore complex.

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In consequence, proapoptotic factors are released from the inner mitochondrial membrane into the cytosol, such as cytochrome c which contributes to the formation of the apoptosome and the subsequent activation of the caspase cascade.

The P53 Network- Survival and Cell Death Regulation

In a normal growing viable cell, the p53protein is in a metastable state, i.e. p53 is susceptible to targeted ubiquitination and subsequent proteasomal degradation. Mdm2 directly interacts with p53 and thereby catalyzes ubiquitination ofp53. Ubiquitination of p53 can be reversed by the action of the deubiquitinating enzyme HAUSP which thereby can rescue p53 from degradation. P53 is stabilized in response to genotoxic stress such as DNA damage which leads to the phosphorylation of p53 at several specific serine and threonine residues. Stabilized and activated p53 can translocate into the nucleus where it activates the transcription of proapoptotic genes and suppresses the transcription of anti-apoptotic genes what under certain conditions can result in the induction of apoptosis. P53-mediated apoptosis signaling is dependent on the interplay of many regulatory factors, including proto oncogenes as well as tumor-suppressors.

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Mdm2 activity is positively regulated by the action of the Akt kinase: when Mdm2 is phophorylated by Akt, Mdm2 is able to translocate from the cytosol to the nucleus where it unfolds its inhibitory effect on p53. Akt kinase, on the other hand, is activated in response to survival signals coming from growth factor receptors. This is therefore an instructive example for the negative regulation of proapoptotic, p53mediated signals by survival signaling. Whereas Akt kinase positively regulates Mdm2 activity, Mdm2-mediated suppression of p53 is blocked by the action of the ARF tumor suppressor. By binding to Mdm2, ARF prevents the interaction between Mdm2 and p53 and therefore stabilizes and activates p53. ARF expression is dependent on the transcription factor E2F-1 which is regulated by the retinoblastoma (Rb) tumor-suppressor and by the action of oncogenes. As an example, mitogenic signals lead to the activation of oncogenes such as c-myc and ras which among others activate E2F-1, resulting in increased ARF activity, stabilization of p53 and induction of apoptosis. Therefore, increased mitogenic signaling or inappropriate oncogenic activity not necessarily causes excessive proliferation but in cells with intact p53 signaling pathways can act as apoptosis inducers.

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