Apoptosis
• Cells receive external signals from
the
surrounding
environment,
which cause a cellular response
such
as
differentiation,
proliferation, secretion, etc. One
appropriate response to a signal is
for the cell to commit suicide presumably for the good
of the organism.
• Cells can die in either of two ways.
First, due to physical or chemical injury
or to membrane damage, which leads
to necrosis or cell disintegration. The
dead tissue is then taken up and
degraded
by
phagocytic
cells.
Second,
programmed cell death,
also referred to as apoptosis.
• Apoptosis:
Apoptosis is a cell death process which
occurs during development and aging
of cells. It is also induced by cytotoxic
lymphocytes (CTL), anti-cancer drugs,
g- or UV-irradiation, a group of
cytokines called
death factors, and
deprivation of survival factors.
Apoptosis
• Apoptosis is mediated
by a family of cysteineaspases
(caspases),
which are expressed as
inactive zymogens and
are
proteolytically
processed to an active
state
following
an
apoptotic stimulus.
• These caspases form a cascade of
proteases which are activated in this
process. They are endoproteases have an
active site Cys (C) and cleave at the Cterminal side of Asp residues (asp) and
hence are known as caspases - cys
containing-asp specific proteases).
• Two pathways leading to
caspase activation
have been characterized,
the extrinsic and
the intrinsic pathways.
Extrinsic pathway:
• The extrinsic pathway is
initiated
by
ligation
of
transmembrane
death
receptors
(CD95,
TNF
receptor, and TRAIL receptor)
to
activate
membraneproximal (activator) caspases
(caspase-8 and -10), which in
turn cleave and activate
(effector) caspases such as
caspase-3 and -7.
Extrinsic pathway:
• This pathway can be
regulated by c-FLIP, which
inhibits upstream activator
caspases, and inhibitor of
apoptosis proteins (IAPs),
which
affect
both
activator
and
effector
caspases.
Intrinsic pathway:
• The
intrinsic
pathway
requires disruption of the
mitochondrial membrane
and
the
release
of
mitochondrial
proteins
including
Smac/DIABLO,
HtRA2, and cytochrome c.
Cytochrome c functions
with Apaf-1 to induce
activation of caspase-9.
• Mitochondrial
membrane
permeabilization is regulated by the
opposing
actions
of
proand
antiapoptotic Bcl-2 family members.
Multidomain proapoptotic Bcl-2 proteins
(e.g., Bak and Bax) can be activated
directly following interaction with the
BH3-only Bcl-2 protein Bid.
• Alternatively, binding of other
BH3-only proteins (e.g., Bad,
and Bim) to antiapoptotic
Bcl-2 proteins (e.g., Bcl-2
and Bcl-XL).
• Intrinsic stresses such as oncoproteins,
direct DNA damage, hypoxia, and
survival factor deprivation, can activate
the intrinsic apoptotic pathway. p53 is a
sensor of cellular stress and is a critical
activator of the intrinsic pathway. The
DNA checkpoint proteins, directly
phosphorylate and stabilize p53.
• p53 can initiate apoptosis
by transcriptionally
activating proapoptotic Bcl-2
family members (e.g., Bax, Bak)
and repressing antiapoptotic
Bcl-2 proteins (Bcl-2, Bcl-XL)
and IAPs.
• Characteristics:
Apoptosis was initially characterized by
morphological changes of dying cells.
During apoptosis cells shrink, and microvilli
on the plasma membrane disappear. The
nucleus
is
also
condensed,
and
fragmented; at the final stage of apoptosis
the cells themselves are fragmented with
all cellular contents inside; one
of the biochemical hallmarks
of apoptosis is the fragmentation
of chromosomal DNA into
nucleosome size units (180 bp).
Clinical applications:
• Disruption of intrinsic apoptotic
pathway is extremely common
in cancer cells.
• The p53 tumor suppressor gene
is the most frequently mutated
gene in human tumors, and loss
of p53 function can both
disable
apoptosis
and
accelerate tumor development
• It has also been observed that
mutations or of upstream
regulators of Bcl-2 proteins are
associated with cancer.
• Mutations in the p53 gene or
in the p53 pathway can
produce multidrug resistance
in vitro and in vivo, and
reintroduction of wild-type
p53
can
re-establish
chemosensitivity.
or
altered
• Mutations
expression of Bcl-2-related
proteins have also shown
relation
to
multidrug
resistance in human cancers.
Unregulated apoptosis could exacerbate or
cause disease such as:
• AIDS, in which T helper cell numbers plummet. Part of
the dramatic decline in these cells might be caused
by health T helper cells being tricked into committing
suicide;
• neurodegenerative diseases like Alzheimers;
• ischemic stroke, when restricted blood flow to certain
regions of the brain can lead to neural death through
increased apoptosis'
• cancer, in which tumor cells lose
their ability to undergo apoptosis;
• autoimmune disease, in which
self-reactive immune cells trick
normal body cells to kill themselves;
• viral disease;
• Apoptosis is very different from tissue
necrosis caused by an acute injury, in
which cells swell and burst (from osmotic
pressure differences) and cause a
significant immune response.
• Apoptosis consists of 4 steps:
1-the decision to activate the pathway;
2-the actually "suicide" of the cell;
3-engulfment of the cell remains
by specialized immune cells
called phagocytes;
4-degradation of engulfed cell.
• Apoptosis does not require
new
transcription
or
translation, suggesting that
the molecular machinery
required for cell death lay
dormant in the cell, and
just requires appropriate
activation. What "signals"
induce apoptosis?
Detection of apoptosis
Apoptotic cells can be recognized by:
• Electron microscope.
• Staining of the condensed nuclei with
fluorescence dyes Hoechst or DAPI.
• Apoptotic cells expose phosphatidyl-serine
to the cell surface, which can be stained
with fluorescence labelled annexin V.
• The fragmented DNA can be
detected by TUNEL procedure.
• Or by electrophoresis of the isolated
DNA on an agarose gel, which
yields a ladder of DNA fragments
with a unit size of 180 bp.
Any Questions ?
• The other effector molecule in apoptosis is Apaf-1
(apoptotic protease activating factor), which,
together with cytochrome c, recruits pro-caspase 9
in an ATP (or dATP)-dependent manner, and
stimulates processing of it to mature enzyme.
• The other regulators of apoptosis are the Bcl-2 family
members. Eighteen members have been identified
for the Bcl-2 family, and divided into three
subgroups based on their structure. Members of the
first subgroup, represented by Bcl-2 and Bcl-xL, have
an anti-apoptotic function. Members of the second
subgroup, represented by Bax and Bak, as well as
members of the third subgroup such as Bid and Bad,
are pro-apoptotic molecules.
• The signal transduction pathway for a death factor
(Fas ligand)-induced apoptosis has been well
elucidated. Binding of Fas ligand to its receptor
results in the formation of a complex (disc, deathinducing signaling complex) consisting of Fas, FADD,
and pro-caspase 8. Pro-caspase 8 is processed to
an active enzyme at the disc. There are two
pathways downstream of caspase 8. In one type of
cells, such as thymocytes and fibroblasts, caspase 8
directly activates caspase 3. In type II cells such as
hepatocytes, caspase 8 cleaves Bid, a member of
the Bcl-2 family. The truncated Bid then translocates
to mitochondria and stimulates release of
cytochrome c, which activates caspase 9 together
with Apaf-1. The activated caspase 9 causes
processing of pro-caspase 3 to the mature enzyme
downstream.
Cells can be instructed to undergo apoptosis through cell
surface interactions with other cells which are often
immune cells. One of the jobs of the immune cell is to
destroy an altered cell (for example a virally-infected cell
or a tumor cell).
Immune cells themselves must also die after they are
activated in an immune response.
Activated
lymphocytes (like cytotoxic T cells or natural killer cells)
can target and kill cells using several ways which can
involved apoptosis.
In one, an activated lymphocyte binds to a target cell (like
a virally infected cell) and secrete perforin, a protein
which assemble in the target cell membrane to form a
transmembrane channel.
Other proteins released by the activated lymphocyte can
enter the target cell through the pore and initiate
apoptosis. One such protein that enters, granzyme B is a
protease which activates caspases in the target cell.
THE PERFORIN/GRANZYME
PATHWAY
• When a cytotoxic T cell recognizes antigen on the
surface of a target cell, it releases the contents of its
lytic granules through a calcium-dependent process.
These granules contain two major classes of cytotoxic
effector proteins- perforin and proteases known as
granzymes. Perforin is released through exocytosis at
the point of contact and polymerizes within the
membrane of the target cell, producing a cylindrical
structure in the lipid bilayer that is lipophilic on the
outside and hydrophilic down the length of its hollow
center. Water and salts are then able to enter the cell
through these pores, destroying the integrity of the
target cell membrane.
• In addition granzyme A and B are introduced into the
target cell and activate the caspase family of
proteases.