Cell death can occur through necrosis or apoptosis. Apoptosis is programmed cell death that occurs normally during development and aging. It is triggered through intrinsic or extrinsic pathways. The intrinsic pathway involves mitochondria releasing cytochrome c which activates caspases like caspase-9. The extrinsic pathway involves death receptors activating caspase-8. Caspase-8 then activates caspase-3 through Bid cleavage or JNK signaling. Cytotoxic T-cells use granzymes like granzyme B and perforin to induce apoptosis through caspase-3. Apoptosis is tightly regulated by both pro-apoptotic and anti-apoptotic genes to control cell death.

1. Dr. Madhuri Kaushish Lily
Associate Professor, Department of
Biotechnology & Microbiology
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2. Apoptosis or Programmed Cell Death
Cell death can occur by either of the two distinct mechanisms,
necrosis and apoptosis. In addition, certain chemical compounds
and cells are said to be cytotoxic to the cell, that is, to cause its
death.
Necrosis (“accidental” cell death): It is the pathological process
which occurs when the cells are exposed to a serious phsical or
chemical insult.
Apoptosis (normal or “programmed” cell death): It is the
physiological process in which a cell brings about its own death
and lysis signaled from outside or programmed in its genes, by
systematically degrading its own macromolecules to eliminate
unwanted or useless cells during development and other normal
biological processes.
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3. The components of the pathway may be present in many or all higher
eukaryotic cells.
During development of a multicellular eukaryotic organism, some cells are
required to die. Unwanted eukaryotic cells are eliminated during
embryogenesis, metamorphosis and tissue turnover. This process provides a
crucial control over the total cell number.
In C. elegans, in which somatic cell lineages have been completely defined,
131 of the 1090 somatic cells formed during adult development undergo
programmed cell death. Cells die predictably at a defined time and place in
each animal.
Cell death occurs during vertebrate development, the most prominent
locations are the immune system and nervous system.
The proper control of apoptosis is crucial in probably all higher eukaryotes.
Failure to apoptose allows tumorigenic cells to survive and thus contributes
to cancer. Inappropriate activation of apoptosis is involved in
neurodegenerative diseases.
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4. Apoptosis involves the activation of a
pathway that leads to suicide of the cell by a
characteristic process in which:
The cell becomes more compact.
Blebbing occurs at the membrane.
Chromatin becomes condensed.
DNA is fragmented
Ultimately dead cells become fragmented
into membrane bound pieces and may be
engulfed by the surrounding cells.
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7. Apoptosis can be triggered by a variety of stimuli,
including:
1.γ irradiation
2.withdrawal of essential growth factor
3.treatment with glucocorticoids
4.ligand activation of some receptors
5.attack of cytotoxic lymphocyte on target cell
6.requires p53, which is a tumor suppressor and act against
cancer
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8. Apoptosis is important, therefore not only in tissue development but in the immune defense
and in the elimination of cancerous cells.
 3 pathways for triggering apoptosis are as follows:
 1. Classical or Common pathway
 2. JNK- Mediated Pathway
 3. Cytotoxic T-Lymphocyte Mediated Pathway
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 1. Classical or Common Pathway:
 It involves the ligand-receptor interaction that triggers activation of a protease which leads to
release of the cytochrome-c from the mitochondria which in turn activates a series of
proteases, whose actions culminate in the destruction of the cell structure.
 A complex containing several components forms at the receptor.
 TNF receptor binds a protein called TRADD which in turn binds a protein called FADD.
 Fas receptor also binds FADD.
 In either case, FADD binds the protein Caspase-8 (also known as FLICE) which has a death
domain as well as protease catalytic activity and which may then trigger a common pathway.
 Members of the caspase family (cysteine aspartate proteases) are important downstream
components of the pathway.
 All caspases are inhibited by crm A (a product of cowpox virus).
 Caspases have a catalytic cysteine and cleave their target at an aspartate.
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9.  The event that activates a caspase is usually an oligomerization.
The trigger to activate the first caspase in the pathway (caspase- 8) is probably an oligomerization
caused by association with the receptor.
The mechanism of activation is to cleave a proenzyme.
 Caspase- 3 is activated by cleaving the pro-caspase precursor to release two fragments that then
associate to form an active heterodimer.
Changes in mitochondria occur during apoptosis and also during other froms of cell death. These
are typically detected by changes in permeability.
In association with these changes, cytochrome-c is released form mitochondria. Its role is to
provide a cofactor that is needed to activate caspase-9.
To move the pathway from the plasma membrane to the mitochondrion, caspase-8 cleaves a
protein called Bid, releasing the C-terminal domain which then translocates to the mitochoindrial
membrane.
This causes cytochrome–c to be released. Cytochrome-c triggers the interaction of the cytosolic
protein Apaf-1 with caspase-9.
When Apaf-1 oligomerizes with procaspase-9, this causes the autocleavage that activates caspase-
9.
Caspase-9 in turn cleaves pro-caspase-3 to generate caspase-3 which is the bets characterized
component of the downstream pathway.
The release of cytochrome-c is a crucial control point in the pathway.
Bid is a member of the important Bcl2 family. Some members of this family are required for
apoptosis while other counteract it.
Bcl2 inhibits apoptosis in many cells.
It has a C-terminal membrane anchor and is found on the outer mitochondrial, nuclear and ER
membranes.
 It prevents the release of cytochrome-c and thus in some way it counteracts the action of Bid.
Caspase-3 finally acts at the effector stage of the pathway.
One known target is the enzyme PARP (Poly [ADP-ribose] polymerase). Its degradation is not
essential but is a useful diagnostic for apoptosis.
Caspase-3 cleaves PARP. Caspase-3 cleaves one subunit of a dimer called DFF (DNA
fragmentation factor).
The other subunit then activates a nuclease that degrades DNA.
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11. 2. JNK-Mediated Pathway:
This is the alternative pathway for triggering apoptosis that
does not pass through Apaf-1 and caspase-9 and which is not
inhibited by Bcl2 and it involves the activation of JNK.
This pathway explains the variable ability of cells to resist
apoptosis in response to Bcl2.
Fas can also activate apoptosis by a pathway that involves the
kinase JNK whose most prominent substrate is the transcription
factor c-Jun.
This leads by undefined means to the activation of proteases.
This pathway is mediated by the protein Daxx (which does not
have a death domain).
Binding of FADD and Daxx to Fas is independent: each adaptor
recognizes a different site on Fas.
The 2 pathways function independently after the Fas has engaged
the adaptor.
 TNF receptor can also activate JNK by means of distinct adaptor
proteins.
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13. 3. Cytotoxic T-Lymphocyte Mediated Pathway:
It is another apoptotic pathway that is triggered by cytotoxic
T-lymphocyte which kill target cells by a process that
involves the release of granules containing serine
proteases and other lytic components.
One such component is perforin which can make holes in
the target cell membrane and under some conditions can kill
target cell membrane.
The serine proteases in the granules are called granzymes.
In the presence of perforin, grnazyme B can induce many of
the features of apoptosis including fragmentation of DNA.
It activates a caspase called Ich-3 which is necessary for
apoptosis in this pathway.
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15. Control of Apoptosis
It involves components that inhibit the pathway as well as
those that activate it.
Mutations in ced-3 and ced-4 cause the survival of cells
that usually die demonstrating that these genes are
essential for cell death.
Ced-3 codes for the protease activity. Ced-4 codes for
the homolog to Apaf-1.
Ced-9 inhibits apoptosis. It codes for the counterpart of
Bcl2. A mutation that inactivates ced-9 is lethal because
it causes the death of cells that should survive.
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