APOPTOSIS TUTORIAL NOTES

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What is Apoptosis?
In any given multicellular organism, the cells form a highly complex and organised
community. The number of cells in the communities is partially regulated by the rate of cell
division. However another important control is the rate of cell death. Any cells no longer
required in a community undergo a suicide process via an intracellular death program. This
process of cell suicide is commonly referred to as Apoptosis, a Greek derived word meaning
"falling off".
Apoptosis was formerly knows as Programmed cell death (PCD), and this was attributed to
instances where sequences of events in a cell would lead to its death. Expanding on this it
became noted that protein synthesis was a requirement in initiating cell death in
developmental situations.
Apoptosis is not the only form of cell death, a less coordinated form of cell death exists,
known as necrosis. There are marked differences between necrotic and apoptotic processes.
Necrosis is often viewed as death due to cell damage as a result of extreme variance from
physiological conditions. Such variations include hypothermia and hypoxia. Direct plasma
membrane damage can also be caused by lytic viruses. Homeostasis is lost in necrotic cells
and as a result of water and ion influx, intracellular organelles and the cell itself swell and
rupture. This lysis event empties cytoplasmic content into extracellular regions causing tissue
damage and an inflammatory response.
Apoptosis and Necrosis
The below table summarises many of the differences between Apoptosis and Necrosis and
categorises them as morphological, biochemical or physiological features.

APOPTOSIS

NECROSIS
Morphological Features

Membrane blebbing, no loss

of integrity.

Aggregation of chromatin at
the nuclear membrane.

Shrinking of cytoplasm and

condensation of nucleus.

Loss of membrane integrity.

Swelling of cytoplasm and mitochondria.

Total cell lysis.

No vesicle formation, complete lysis.

Fragmentation of cell into

smaller bodies.

Membrane bound vesicles

form as apoptotic bodies.

Mitochondrial leaking due to

pore formation by bcl-2
family of proteins.

Disintegration of cellular organelles.

Biochemical Features

Tightly regulated involving

activation and enzymatic
steps.

Loss of regulation of ion homeostasis.

Energy independent.

Energy (ATP) dependant.

Random DNA digestion.

Non-random fragmentation
of DNA.

Postlytic DNA fragmentation.

Prelytic DNA fragmentation.

Activation of caspase
cascade.
Physiological Features

Affects individual cells.

Affects groups of cells.

Induced by physiological
stimuli (lack of growth
factors for example).

Caused by non-physiological disturbances

(hypoxia, lytic viruses metabolic poisons
etc)

Phagocytosis by adjacent
cells or macrophages.

Phagocytosis by macrophages.

Significant inflammatory response.

No inflammatory response.

Apoptosis and Cancer

It is recognised that one of the contributors to cancer development is a lack of apoptosis, as

should cells lose control of death then there is less restraint on tumour proliferation.
Decreased apoptosis can be linked to several forms of cancer.
Why does Apoptosis occur?
There are varying reasons Apoptosis occurs; three important examples are during "normal"
development of an organism, to combat any of our cells which pose a threat to the self, and in
response to DNA damage. Each of these three examples is discussed below.

"Normal" development
Apoptosis is an important feature of normal development. For example in amphibians and
experimentally Xenopus Laevis, apoptosis is linked to regression of the tadpole tail,
remodelling of the intestine, and remodelling of the skin from a thin larval state to a striated
adult skin when adapting to a dry environment. Similarly the development of toes and fingers
on limbs has an element of reliance on apoptosis. Apoptotic degradation of tissue allows for
the separation and normal development of fingers and toes. Apoptosis is one of the factors
associated with degradation of the uterus lining in several mammals during menstruation and
apoptotic cells also accumulate in the ovary prior to ovulation as part of a process called
atresia.

Threatening cells
Maybe a more obvious reason for apoptotic processes is to remove cells which pose a threat
to the body, in a controlled manner. Cytotoxic T cells (CD8 cells) of the immune system
destroy target cells via apoptosis. This is important as the viral content of an infected cell is
not dangerously released into the surrounding cell environment. CD8 cells initiate apoptosis
by releasing cytotoxins such as perforin. This forms pores in a cell membrane to
accommodate granzymes (serine proteases) which cleave cell proteins to activate nucleases
and enzymatic initiators of apoptosis. Alternatively Fas ligands on CD8 cells can bind with a
target cell receptor and trigger a signalling cascade for apoptosis. This is also a useful method
of controlling lymphocyte population.

DNA damage
In DNA damaged cells, apoptosis occurs to remove cells which may contribute to improper
development or to prevent cells from becoming cancerous. Cells falling into such criteria are
prevented from continuing through the cell-cycle by a tumour suppressor gene known as p53.
The p53 gene is considered the "guardian of the genome" and should DNA damage be
irreparable, p53 activates "suicide" genes to initiate apoptosis and remove the threatening

cell.
Inducing Apoptosis
In the last section we saw examples of situations where Apoptosis is called upon and why it
is an important attribute to the body. This section on inducing Apoptosis expands on this by
exploring how Apoptosis can be initiated. Inducing Apoptosis is closely linked to the
mechanisms of Apoptosis therefore this section and the next have relation to one another. The
content on inducing Apoptosis here could be viewed as optional detail, however for those
with an interest it may provide a greater understanding when you explore the actual
mechanisms by which Apoptosis occurs.
Apoptosis occurrence can be categorised broadly into two main categories; As a result of a
cell losing positive-survival signals or due to the cell being exposed to negative signals.
Loss of positive signals
Growth factors allow cells to continue to develop through the cell cycle. Deprivation of
growth factors can trigger cell death and apoptosis. For example, sympathetic neurons
require nerve growth factor (NGF) to sustain growth. Those neuronal cells which are not
stimulated to a necessary level by NGF display characteristics of cells undergoing apoptosis.
This event is also linked to normal development with approximately half of neurons
committing to apoptosis due to insufficient NGF stimulation from post-synaptic targets.
A further example of growth factor dependence is Interleukin 2-dependent T cells. These
cells do not enter quiescence (inactive or resting period of the cell cycle, G0). Instead, a lack
of IL-2 causes arrest at the G1 period of growth. In this state DNA fragmentation occurs and
apoptosis proceeds.
Receipt of negative signals
Receipt of negative signals evokes a similar effect to withdrawal of positive signals such as
the growth factors discussed. Oxidative stress has been observed to induce apoptosis in
cardio myocyte (muscle) cells in the heart. Oxidative stress occurs during an imbalance of
free radicals and antioxidants in the body systems. Exposure to UV radiation and ionisation
can generate reactive oxygen species (ROS) such as H2O2 and OH. Both the ROS and
action of UV radiation itself on DNA can infer damage which, if not repaired, can promote
apoptosis via activation of the tumour suppressor p53.
Death activators are molecules which bind to a cell surface receptor and can trigger apoptosis
and are very important "negative" signals in inducing apoptotic processes. More can be read
about this in the extrinsic pathway discussion in the mechanisms section of the tutorial.
Mechanisms of Apoptosis

The three topics covered in this section of the tutorial are associated with the overall
mechanisms of Apoptosis, the cascades of events which occur to cause Apoptosis. This
includes the intrinsic (mitochondrial) pathway, the extrinsic (death receptor) pathway, and
finally the action of Apoptosis Inducing Factor (AIF).
Mechanisms : Intrinsic
Mitochondria contain a cocktail of proteins which contribute to apoptosis. The intrinsic
pathway is utilised notably in response to DNA damage and extracellular conditions exerting
stress on the cell such as that of ROS. Cytochrome c is a major pro-apoptotic protein released
by the mitochondria as it is essential in activating caspase-9 (previously discussed). In a
normal healthy cell it is maintained within the mitochondria as cytochrome c cannot be
released providing the barrier function of the outer mitochondrial membrane remains intact.
Furthermore the mitochondria also release apoptosis inducing factor to act in a caspase
independent manner.
The release of mitochondrial contents depends on a battle between pro- and anti-apoptotic
members of the Bcl-2 family of proteins. Anti-apoptotic members include Bcl-2 and Bcl-xL,
pro-apoptotic members include Bid and Bax. The anti-apoptotic members are categorised as
Group I in the Bcl-2 family and contain four conserved homology domains, BH1-BH4. Bax
and Bid fall into Group II and III respectively and do not maintain the same level of
homology but both lack the BH4 domain but contain the BH3 domain.
Bcl-2 family members regulate the opening of a permeability transition pore (PTP) though
the exact mechanism of this is not entirely clear. It could be a combination of autonomous
pore formation by Bcl-2 family members or association with PTP complex (PTPC). The
PTPC is comprised primarily of an adenosine-nucleotide translocator found in the inner
mitochondrial membrane, and a voltage dependant ion channel (VDAC) in the outer
membrane. Pro-apoptotic protein release and mitochondrial homeostasis could be governed
by such a pore.
To enhance the apoptotic commitment smac/DIABLO is also released from the mitochondria.
Smac stands for "second mitochondria derived activator of caspase", and DIABLO for
"direct IAP-binding protein with low pl". From their names alone it is quite apparent their
activity. By DIABLO antagonising inhibitor of apoptosis proteins the maturation of
procaspase-3 is no longer blocked.
Mechanisms : Extrinsic
The extrinsic apoptotic pathway is initiated by Fas ligand and TNF family ligand binding to
cell surface receptors.

Fas ligand (FasL); also known as CD95

The combining of FasL with Fas receptors promotes the recruitment of an adaptor
protein known as Fas-associated death domain protein or FADD. FADD contains a
death effector domain (DED) which can associate with, and initiate the activation of
caspases. The complex formed (FasL, Fas, FADD and the caspase) is what constitutes
the death inducing signalling complex. (See Caspase activation section)

Tumour Necrosis Factor-alpha (TNF-)

and Lymphotoxin (TNF-)
These members of the TNF super-family are capable of administrating an apoptotic
signal to cells which possess a corresponding receptor. Receptors for the ligands
possess an intracellular domain known as a "death domain". The process is similar to
that of FasL discussed previously. In the case of TNF the associated death domain is
TRADD (tumour receptor-associated death domain). TRADD can either associate
with the death domain of FADD to activate caspase-8 or it can initiate NF-k and
JNK pathways. The latter promotes an inflammatory response in cells.
Mechanisms : AIF
AIF is a mitochondrial factor implicated in apoptosis. Mammalian AIF is a 67kDa protein
containing a flavin-adenine-dinucletide binding domain. Apoptotic stimuli cause the
translocation of AIF to the nucleus from the mitochondria.
Though AIF acts in tandem with other pro-apoptotic factors it behaves independently of
caspases. AIF causes DNA fragmentation and chromatin condensation in the nucleus by
possibly recruiting proteases and nucleases, though this is not entirely clear.
Caspases
The changes observed in apoptotic cells are largely accounted for by the action of caspases.
Caspases are a large family of proteins and specifically are cysteine proteases. The name
"caspases" comes from Cysteinyl-Aspartate-Specific-ProteASES. A protease is an enzyme
that digests protein by peptide-bond hydrolysis. Caspases are synthesised as pro-caspases that
are processed by proteolytic cleavage at specific aspartate residues to become active. This
means pro-caspases are zymogens (inactive enzyme precursors).
Each of the pro-caspases contains a highly homologous protease domain and an N-terminal
pro-domain. The pro-domain is followed by a large 20kDa subunit and a smaller 10kDa
subunit known as p20 and p10 respectively. Depending on pro-domain structure, and
function, the caspases are categorised into 3 main groups.

Large pro-domains give inflammatory caspases (Group I) and initiator of apoptosis

caspases (Group II)

Short pro-domains give effector caspases (Group III)

The first caspase identified was ICE (interleukin-1-converting enzyme) also known as
caspase-1. Though identified in humans the actual involvement of caspases in apoptosis was
uncovered in Caenorhabditis elegans (nematode worm). In the nematode worm a ced-3 gene
was noted to encode a cysteine protease baring close resemblance to mammalian ICE. Of the
14 mammalian caspases identified, 11 are found in humans and 7 are well known to have
important roles in apoptotic processes.
Caspase activation
In mammals it is caspase-2, -8, -9 and -10 which are considered to be the initiator caspases.
Caspase-3, -6 and -7 are considered to be the effector caspases. Shown below is a schematic
representation of the mentioned caspases. The prodomains of initiator caspases contain
homotypic interaction motifs, such as the caspase-recruitment domain (CARD) and deatheffector domain (DED). Four surface loops represented by L1-L4 shape the catalytic groove.
The red line seen at the start of L2 is the site of the catalytic residue Cys.
The activation of the effector caspases can be categorised into three main pathways. One
means of activation occurs due to upstream initiator caspases which cleave the effector
caspases at Asp (aspartate) residues separating the p20 and p10 subunits. This is the major
means of caspase-3, -6 and -7 activation but it does not explain how the upstream initiator
caspases become active.
There are at least two possible initiator activation protocols; induced proximity, and
association with a regulatory subunit.
Induced proximity activation
The first of the two ativation models to be discussed is that of induced proximity, where the
close arrangement of pro-caspases is sufficient to initiate their activation.
Following ligand binding at the cell surface, death receptors such as CD95 (see Inducing
Apoptosis section) aggregate to give membrane bound signalling complexes. This causes
procaspase-8 recruitment via adaptor proteins (FADD). The high concentration of
procaspase-8 in close proximity results in sufficient enzymatic activity to cause mutual
cleavage and thus activation to caspase-8. However, there is an element of controversy
regarding the induced proximity model.
The complex formed at the cell membrane is a death-inducing-signalling-complex or DISC.
An updated proposal to the induced proximity idea is that of Proximity-induceddimerisation.This theory by Boatright et al does not ignore the original findings of induced

proximity activation spawned in 1998, but alludes to a level of control expected in complex
biological processes. Rather than simply clustering and self activating it is thought that
caspase-8 monomers dimerise at the DISC and the cleavage is a stabilising event. After
dimerisation the N-terminal DED domain is removed by the DISC and the active caspase is
released into the cytosol.
Association with a regulatory subunit
The second means of initiator caspase activation is the most complex and is associated with
caspase-9.
Proteolytic processing of procaspase-9 has limited effect on its activity. It is only when
associated with a protein cofactor called Apaf-1 that it becomes fully active. Apaf-1 and
cytochrome c are both needed for caspase-9 activation. Cytochrome c is released from the
mitochondria of pre-apoptotic cells and will bind with Apaf-1 in the presence of ATP. A
conformational change in Apaf-1 allows several molecules to associate with one another.
7 Apaf-1 molecules form a wheel like structure with cytochrome c. A caspase recruitment
domain (CARD) is present on pro-caspase 9 and Apaf-1. The two sets of CARD domains
interact forming an apoptosome. The pro-caspase 9 is then cleaved and its mature active form
can go on to activate effector caspases.