At the end of this CAL you will be able to:

  • Understand  that there are different types of cell death – apoptosis and necrosis
  • Describe situations in which apoptosis occurs
  • Describe how apoptosis is controlled
  • Describe the morphological and biochemical changes involved in apoptosis

Apoptosis v necrosis Part 1 of 10

There are two main types of cell death, apoptosis and necrosis.

Apoptosis is usually part of a regulated process, and has been called ‘programmed cell death’ or ‘cell suicide’. It is a carefully regulated event, requiring energy from the dying cell, usually resulting in cell shrinkage and fragmentation. Phagocytosis of the resultant apoptotic bodies ensures there is no associated inflammation and bystander tissue damage.

Necrosis typically results from a significant cellular injury. Cells swell and burst, releasing intracellular contents in an uncontrolled manner. This causes inflammation and tissue damage.

Physiological apoptosis Part 2 of 10

Apoptosis can be –

  • Physiological, occurring as part of normal development or function
  • Pathological, as part of a disease process

Physiological apoptosis

Apoptosis can occur as a normal physiological process during development or adult tissue homeostasis.

Developmental apoptosis

In development, unwanted cell populations are removed by apoptosis.

One example of this in human embryological development is the removal of interdigital tissue to form defined digits. Dying cells are identified in these images as red, and are removed during development to leave well-defined digits.

©M Suzanne, H Steller 2013 CC BY-NC-ND 3.0
A schematic representation of a developing limb is shown with apoptosis indicated in red (up). When cells from the interdigital zones are removed, a new shape is revealed (down)

Apoptosis in adult homeostasis

In adult homeostasis, many cell populations are constantly being replaced and refreshed. Often the removal of the old, unwanted population is by apoptosis.

An example is the epithelium of intestinal mucosa.

©Tim Kendall, University of Edinburgh 2018 CC BY-SA
Histology of normal colonic mucosa.

Cells derive from progenitor cells at the base of crypts and move up the crypts (and villi, in small intestinal mucosa) as older cells are removed by apoptosis from the lumenal aspect or villous tip.

This image demonstrates the proliferating epithelial cells restricted to the deep mucosa using an antibody recognising Ki-67. Ki-67 is a cellular protein expressed by proliferating cells at all stages of the cell cycle, but not expressed by cells in the resting phase (G0).

©Laure Droy-Dupré et al, Disease Models & Mechanisms 2012 5: 107-114 CC-BY license
Ki67 immunopositive cells within crypt bases

In contrast, apoptotic epithelial cells are restricted to the lumenal aspect.

©(2005) National Academy of Sciences http://www.pnas.org/page/about/rights-permissions
Apoptotic (TUNEL positive) epithelial cells on the lumenal surface of colonic mucosa

Pathological apoptosis Part 3 of 10

Cells can undergo apoptosis when damaged or involved in a disease process.

For example, in viral hepatitis, some hepatocytes (liver epithelial cells) infected by virus undergo apoptosis, often induced by the action of T cells. Apoptotic hepatocytes can often be identified by their characteristic rounded, shrunken shape.

©Tim Kendall, University of Edinburgh 2018 CC BY-SA
An apoptotic hepatocyte (acidophil body, arrowed) in chronic Hepatitis C virus infection.

Regulation of apoptosis Part 4 of 10

Apoptosis is tightly controlled. It can be triggered by the activation of one of two pathways, mitochondrial (intrinsic) and death-receptor mediated (extrinsic). There is some cross-talk between the two, but both lead to a final common pathway.

©Tsgupta [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)], from Wikimedia Commons
Common effector caspases are activated by separate initiator caspases of both the extrinsic and intrinsic apoptosis pathways.

Mitochondrial pathway

A cell receives signals from all elements of the extracellular environment – other cells, extracellular matrix and soluble factors.

Under homeostatic conditions, these signals lead to the balance of intracellular protein Bcl-2 family members favouring ‘pro-survival’ factors, and the maintenance of intact mitochondrial membranes.

If these ‘survival’ signals are withdrawn, the Bcl-2 family member balance shifts and pro-apoptotic family members become dominant.

This leads to release of Cytochrome c from the mitochondria, leading to activation of caspase 9 and then the final common pathway mediated by effector caspases.

Extrinsic (death-receptor mediated) pathway

Many cells have cell surface receptors that contain intracellular ‘death domains’ for example Fas. Binding of an appropriate ligand with the receptor, for example, FasL, can lead to activation of the effector pathway by a route independent of mitochondrial factors.

Receptor ligation activates signalling through a death-associated signalling complex (DISC). The DISC is a complex of the ligand, receptor, and associated signalling proteins such as FAS-associated death domain protein (FADD), and leads to the activation of caspase 8 and the final common executioner pathway.

Intracellular machinery of apoptosis Part 5 of 10

A class of proteins called caspases mediate initiation of the final common pathway (initiator caspases) or the final events themselves (executioner caspases).

©Tsgupta [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)], from Wikimedia Commons
Initiator Pro-caspases have a prodomain that allows recruitment of other pro-caspases, which subsequently dimerise. Both pro-caspase molecules undergo cleavage by autocatalysis. This leads to removal of the prodomain and cleavage of the linker region between the large and small subunit. A heterotetramer is formed.
©Tsgupta [CC BY-SA 4.0 (https://creativecommons.org/licenses/by-sa/4.0)], from Wikimedia Commons
Executioner caspases constitutively exist as homodimers. The red cuts represent regions where initiator caspases cleave the executioner caspases. The resulting small and large subunit of each Caspase-3 will associate, resulting in a heterotetramer.

Caspases (c-aspases) are cysteine proteases (using the cysteine group for substrate cleavage) and aspases (cleaving C-terminal to aspartic acid residues).

All caspases are present in inactive forms to prevent unwanted cell death, requiring cleavage of prodomains for activation.

The mitochondrial or death-receptor mediated pathways lead to activation of different initiator caspases, e.g. caspase 9 and caspase 8, respectively.

Active initiator caspases cleave executioner caspases, activating them to start the final common pathway that leads to degradation of cellular components.

Control of an important process by multiple layers of activation (cascade) is a common method in biological systems.

Morphology of apoptosis Part 6 of 10

After apoptosis is initiated in a cell, a regular sequence of events takes place. Characteristic morphological changes occur (phase 1) before fragmentation by apoptotic body formation (phase 2) and finally, phagocytosis and resolution (phase 3).

©Sandy Reid, University of Edinburgh 2017 CC BY-SA
Membrane blebbing, apoptotic body formation and final phagocytosis during apoptosis.

Activated executioner caspases lead to characteristic morphological changes in the cytoplasm and nucleus in a regular sequence.

Cytoplasmic changes

Firstly, the cell condenses and the endoplasmic reticulum dilates. The chromatin condenses at the periphery of the nucleus, and then fragments of intracellular components bleb off in plasma membrane-bound packages called apoptotic bodies.

This can be observed in cultured cells with a light microscope.

©Tim Kendall, University of Edinburgh 1999 CC BY-SA
Blebbing of the plasma membrane of an apoptotic cell (serum deprived hepatic stellate cell, cytospin).

More detailed changes to the cell surface in apoptotic cells are apparent with scanning electron microscopy. As cells begin to undergo apoptosis, microvilli are lost there is surface puckering as a consequence of fusion of ER-derived vacuoles with the surface.

©Sandy Reid, University of Edinburgh 2017 CC BY-SA
Loss of surface microvilli and puckering evident on the plasma membrane of a cell undergoing apoptosis.

Nuclear changes

DNA is degraded by endonuclease cleavage between histones, creating fragments of a regular length corresponding to ‘units of histone’.

The initial nuclear changes are associated with chromatin condensation.

©Sandy Reid, University of Edinburgh 2017 CC BY-SA
Transmission electron photomicrograph of normal cells showing (a) dark heterochromatin and (b) paler euchromatin in the nucleus. The nucleolus (c) is also shown.
©Sandy Reid, University of Edinburgh 2017 CC BY-SA
Transmission electron photomicrograph of an apoptotic cell showing marked chromatin condensation at the nuclear periphery (a) and a fragmented nucleolus (b).

The nucleus then fragments as nuclear components, organelles and cytoplasm are packaged into apoptotic bodies.

©Sandy Reid, University of Edinburgh 2017 CC BY-SA
Transmission electron photomicrograph showing the later nuclear features of apoptosis. Separate aggregates of chromatin (a) free within the cytoplasm. Ribosomes (b) are also present, and all elements are packaged as membrane-bound apoptotic bodies.

When the DNA of apoptotic cells is run on a gel to separate DNA fragments based on size, a characteristic nuclear ladder formed by multiples of 200 base pairs (‘unit of histone’). In contrast, the DNA from necrotic cells is degraded haphazardly and appears as a smear when similarly examined.

©Tim Kendall, University of Edinburgh 2018 CC BY-SA
The DNA of apoptotic cells is cleaved in a uniform manner between histones to create a ‘ladder’ when separated on an agarose gel. In contrast, the DNA is cleaved haphazardly during necrosis, producing a ‘smear’.

Resolution Part 7 of 10

Apoptosis does not lead to inflammation in surrounding tissue, meaning there is no ‘bystander’ tissue damage.

To achieve this, cells undergoing apoptosis release soluble factors (e.g. nucleotides ATP and UTP, chemokine CX3CL1) that recruit phagocytes.

Recruited phagocytes recognise apoptotic cells by the presence of ‘eat me’ molecules such as phosphatidylserine present on their surface.

The phagocyte responds to these signals by engulfing the apoptotic cell or body. Degradation of the engulfed component completes the process.

Macrophage clearance of apoptotic bodies
©Ravichandran 2010 CC BY 3.0
Different steps involved in efficient apoptotic cell clearance. The find-me signals (such as low levels of nucleotides ATP and UTP, fractalkine, lysophosphatidylcholine, or sphingosine 1-phosphate) released by apoptotic cells help attract motile phagocytes to the proximity of the cell undergoing apoptosis. The phagocytes then use engulfment receptors on their surface to engage eat-me signals on apoptotic cells. For clarity, only the PtdSer on the apoptotic cells engaged by cognate receptors is depicted. Engagement of the engulfment receptors (linked to PtdSer recognition) has been shown to stimulate release of anti-inflammatory cytokines such as TGF-β, IL-10, and prostaglandin E2 (PGE2). The intracellular signaling induced within the phagocyte by the ligand-receptor interactions leads to cytoskeletal rearrangements and internalization of the dying cell. The phagocyte processes the engulfed corpse through a series of steps, and proper digestion seems to be important for continued uptake of other dying cells by phagocytes.

Failure of apoptosis Part 8 of 10

Failure of normal apoptosis can lead to different sequelae depending on the context.

Developmental failure of apoptosis

Failure of normal deletion of unwanted cell populations during development can produce a range of abnormalities.

For example, failure of the normal apoptotic deletion of interdigital tissue leads to syndactyly ( from the Greek Syn, meaning together, and Dactylos meaning digit). Syndactyly is one of the most common hereditary limb malformations, with a wide range of clinical manifestations and underlying causes.

©AzaToth [CC BY-SA 3.0 (https://creativecommons.org/licenses/by-sa/3.0)], from Wikimedia Commons
Feet with partial simple syndactyly
©CC BY-SA 3.0 from Wikimedia Commons
X-ray of hands with type 1 syndactyly.

Failure of apoptosis in adult homeostasis

Failure of normal adult cell apoptosis required for maintenance of normal tissues also has deleterious effects.

Failure of apoptosis of colonic epithelial cells, as a consequence of acquired mutations in genes responsible for the apoptotic machinery and its regulation, is one of the abnormalities permitting the development of adenomas as part of the neoplastic adenoma-carcinoma sequence.


Copyright © 2011 Michael Bonert ( https://commons.wikimedia.org/wiki/User:Nephron
Adenoma in a large bowel resection.

Acquired abnormalities in apoptosis are usually combined with increased cell proliferation.

©Peng et al.; licensee BioMed Central Ltd. 2013
Large numbers of proliferating (Ki67-positive) cells are seen throughout adenomatous epithelium but very few apoptotic (caspase 3) cells are present, a disruption of the distribution observed in the normal colonic mucosa.

In some tumours, specific abnormalities in apoptotic components are directly related to prognosis; for example, patients with Bcl-2 protein over-expression in lymphoma have a poorer prognosis.

Apoptosis as a therapeutic tool? Part 9 of 10

Given that an imbalance between cell proliferation and death to permit autonomous growth is central to the development of cancer, and that acquired abnormalities hampering apoptosis are a regular finding in cancer, it follows that strategies to promote or induce apoptosis may have therapeutic potential.

Early clinical trials to treat different cancers by promoting apoptosis, for example by blocking IAPs (native Inhibitors Of Apoptosis) or the anti-apoptotic Bcl-2 family of proteins, are underway.

Questions Part 10 of 10