Pathology of apoptosis
1. Outline the sequence of morphological changes that occur in cells
undergoing apoptosis
Apoptosis is a pathway of cell death that is induced by a tightly regulated intracellular
program in which cells destined to die activate enzymes that degrade the cells own
nuclear DNA, as well as nuclear and cytoplasmic proteins. The cells plasma
membrane remains intact, but its structure is altered in such a way that the apoptotic
cell becomes an avid target for phagocytosis. Phagocytosis is required so dead cell
can be rapidly cleared before its contents can be leaked out and damage surrounding
cells.
Morphological changes to a cell undergoing apoptosis include;
- Cell Shrinkage – cell smaller in size, dense cytoplasm, organelles tightly
packed together
- Nuclear condensation – “most characteristic feature of apoptosis” – chromatin
aggregates peripherally under the nuclear membrane into dense masses
- Nuclear Fragmentation – nucleus itself may break up, producing two or more
fragments
- Formation of cytoplasmic blebs and apoptotic bodies – apoptotic cell first
shows surface blebbing, then undergoes fragmentation into membrane bound
apoptotic bodies composed of cytoplasm and organelles, with or without
nuclear fragments
- Phagocytosis of apoptotic cells or cell bodies, usually by macrophages –
Expression of phosphatidylserine on the outer plasma membrane of apoptotic
cells/ apoptotic bodies may express thrombospondin, an adhesive
glycoprotein/ proteins secreted by phagocytes act to opsonize the cells for
phagocytosis. This results in removal of apoptotic cells, with adjacent healthy
cells migrating or proliferating to replace the space occupied by the now
deleted apoptotic cell.
NB. Plasma membrane remains intact during apoptosis, where as plasma membrane
damage occurs during necrosis. This results in cell swelling rather than shrinkage
2. Identify typical apoptotic cells and bodies histologically and in
electron micrographs
The cell demonstrates classical features of apoptosis, with blebbing of the nucleus and
cytoplasm in preparation for phagocytosis of the apoptotic debris by phagocytes and
other surrounding cells.
3. List the circumstances in which cell death by apoptosis might be
expected
Apoptosis occurs normally in many situations, and serves to eliminate unwanted or
potentially harmful cells, or cells no longer required.
Important physiological situations where death by apoptosis may occur include
- Programmed destruction of cells during embryogenesis
- Hormone dependent involution in adults ie endometrial cell breakdown in
menstruation
- Cell deletion in proliferating cell populations ie rapidly dividing cells of GIT
epithelia
- Death of cells that have served there purpose ie neutrophils and lymphocytes
- Elimination of potentially harmful self-reactive lymphocytes ie maturation
- Cell death as a defence mechanism against virus infection mediated by CD8
cells
Pathologic states also are responsible for cell death
- Injurious stimuli ie radiation and cytotoxic chemicals
- Heat, hypoxia, stress
- Viral diseases
- Loss of growth factors or hormones
4. Outline the biochemistry and molecular biology of apoptosis with
particular reference to cell shrinkage and condensation, cell budding,
DNA fragmentation, receptor-mediated phagocytosis, tissue
transglutaminase activity, and the roles of c-myc, p53, bcl-2 and
caspases
Apoptotic cells exhibit a distinct array of biochemical changes that underlie the
structural changes described earlier. Some specific features include;
Protein cleavage
A feature of apoptosis is protein hydrolysis involving the activation of several
cysteine proteases called caspases ( ‘c’ from cysteine protease; ‘aspase’ from the
ability of the enzyme to cleave after aspartic acid residues). There are approximately
10 members of the caspase family and they can be divided into two functional groups;
1. Initiator caspases – cleave inactive pro-forms of effector caspases, thus
activating them. Include caspase-2, -8, -9, -10
2. Effector (executioner) caspases – cleave other protein substrates within the
cell to trigger apoptotic processes. Include caspase-3, -6, -7
Caspases are regulated at the post-translational level, ensuring that they can be rapidly
activated. They exist initially in an inactive form (pro-caspase or zymogen) and must
undergo an activating cleavage for apoptosis to be initiated. The important thing is
that caspases can be activated by other caspases as well as themselves. Once initiator
caspase is cleaved and activated, the death program is set in motion via the rapid and
sequential activation of other (executioner) caspases. Caspases then activate other
cellular proteins, such as Lamins – breakdown nuclear scaffold and cytoskeleton
 DNAses – degrade
These changes underlie the nuclear and cytoplasmic structural alterations seen in
apoptotic cells
DNA breakdown
Characteristic breakdown of DNA into large 50-300kb pieces, and then
internucleosomal cleavage of DNA by endonuclease into oligonucleosomes of 180200bp. Fragments may be visualised on agarose-gel electrophoresis. Endonuclease
activity the basis for detection of apoptosis
Phagocytic Recognition
Apoptotic cells express phosphatidylserine in the outer layers of their plasma
membranes, having flipped out from the inner layers. Also, apoptotic bodies may
express thrombospondin, an adhesive glycoprotein. Other proteins secreted by
phagocytes may also act to opsonize the cells for phagocytosis. These alterations
permit the early recognition of dead cells by macrophages, for quick removal without
further damage and with out the release of pro-inflammatory mediators
MECHANISMS OF APOPTOSIS
Apoptosis is induced by a cascade of molecular events that can be initiated in a
number of distinct ways. The end result of this initiation is the culmination of
activated caspases. The process of apoptosis may be divided into an Initiation Phase,
in which caspases become catalytically active, and an Execution phase, during which
these enzymes act to cause a series of events leading to cell death. Initiation occurs
primarily via signals from two distinct pathways;
1. The Extrinsic (Death Receptor-Initiated) Pathway.
This pathway is initiated via the engagement of cell surface receptors on a variety of
cells. These include the Tumour Necrosis Factor (TNF) and Fas family of receptors.
These receptors contain a protein cytoplasmic domain. Upon engagement by ligand ie
FasL or TNF, receptor molecules aggregate resulting in the formation of a protein rich
cytoplasmic domain known as a Death Domain. This provides a binding site for an
adapter protein that also contains a death domain. For Fas receptors, this is known as
FADD (Fas-Associated Death Domain). For TNF receptors, they associate with
TRADD (TNF Receptor-associated Death Domain), which in turn binds FADD.
FADD attached to death receptors in turn binds inactive pro-caspase-8. Multiple procaspase-8 brought into close proximity with one another cleave one another to
produce active caspase-8 (initiator caspase) which then goes on to activate other
caspases that activate the enzymes that mediate the execution phase of apoptosis.
2. The Intrinsic (Mitochondrial) Pathway
This pathway is related to an increased mitochondrial membrane permeability
resulting in the release of pro-apoptotic molecules into the cytoplasm. One way this
happens is by the withdrawal of growth factors or hormones. In the mitochondria,
growth factors and other survival signals stimulate the production of anti-apoptotic
molecules such as the members of the Bcl family. The Bcl family are a large family of
proteins, some with anti-apoptotic properties and others with pro-apoptotic properties.
The two main anti-apoptotic ones are Bcl-2 and Bcl-X. They reside in the
mitochondrial membrane and are believed to govern the inactivation of mitochondrial
transition pores. When cell is deprived of survival signals or placed under stress,
production of these proteins is lost, and they are replace by pro-apoptotic members
such as Bax, Bak, Bid, Bik etc.
When Bcl-2/-X levels decrease and levels of the others rise, mitochondrial
permeability increases and proteins that activate the caspase cascade leak out. This
includes Cytochrome c (cyt-c). Cyt-c binds to a protein called Apaf-1(Apoptosis
Inducing Factor-1) and this complex activates pro-caspase-9, an executioner caspase.
Bcl-2/-x directly inhibits Apaf-1 activation.
Other mitochondrial proteins that are pro-apoptotic, such as Apoptosis Inducing
Factor (AIF), enter the cytoplasm where they bind to and neutralize Inhibitops or
Apoptosis (IAPs). The normal function of IAPs is to block caspase activation
Other methods of inducing the apoptosis signal to activate caspases,
 DNA Damage Mediated Apoptosis
Exposure of cells to radiation or chemotherapeutic agents induces apoptosis that
involves the tumour suppressor gene p53. p53 accumulates when DNA is
damaged and arrests the cell cycle (at the G1 phase) so repair can take place. If
repair process fails, p53 can trigger apoptosis. It does this through the production
of pro-apoptotic Bcl family proteins (Bax, Bak) and these activate caspases that
cause apoptosis. If p53 is mutated or absent in certain disease states it becomes
incapable of inducing apoptosis favouring cell survival.
 Cytotoxic T-Lympthocyte (CD8) Mediated Apoptosis.
CD8 cells recognise foreign antigens on the surface of infected cells. Perforin is a
cytolytic protein found in the granules of T-cells and NK cells. Upon
degranulation, perforin inserts itself into the target cell's plasma membrane,
forming a pore (Polyperforin channel). Through this pore, a second group of
proteins and enzymes (Granyzymes - B) enter the cytosol and activate caspases
(namely caspase-3, -7). CD8 cells also posses the FasL on their surface, capable of
engaging Fas receptors and inducting Death-Receptor mediated apoptosis
The final phase of Apoptosis, the Execution phase is mediated by a proteolytic
cascade courtesy of activated caspases. Once initiator caspases have been cleaved and
activated from their inactive pro-caspase form, they then go on to activate executioner
caspases. Executioner caspases (which can also be activated directly ie Granzyme B
and p53) then go on to act on many cellular components. They directly cleave
cytoskeletal and nuclear matrix components and activate latent cytoplasmic
endonucleases and DNAses. This results in intracellular degradation, including
fragmentation of nuclear chromatin and breakdown of the cytoskeleton. The end
result is the formation of apoptotic bodies containing intracellular organelles and
other components.
Apoptotic bodies also express new ligands which can be used for recognition and
binding for uptake by phagocytic cells. During early stages of apoptosis, dying cells
secrete chemokine that act to attract phagocytes to the area. Also, apoptotic cells have
marker molecules on their surface which facilitates early recognition and uptake by
phagocytes (ie phosphatidylserine). Phagocytes also secrete substances that bind
specifically to apoptotic cells to opsonise them for phagocytosis. Prompt clearance of
apoptotic cells prevents them from undergoing secondary necrosis and releasing their
cellular contents, having an inflammatory effect.