Apoptosis, autophagy and necrosis

Question	Answer
What is senescence?	The point at which cells no longer divide.
What process in somatic cells is key for senescence?	Telomere shortening.
In healthy adults, cell death usually perfectly balances what?	Cell division - though this cell turnover varies between tissues.
Billions of intestinal epithelial cells die every hour. How often is the entire gut epithelium replaced?	Every 3 to 5 days.
Why is it significant that the cells that undergo the most wear and tear from the environment, such as intestinal epithelial cells, undergo the most rapid turnover?	These cells are subject to the most damage and therefore most at risk of genomic instability, so this protects the genome.
Why is apoptosis important in embryological development?	To remove unnecessary structures that are present as a result of development, such as the interdigital webbing between fingers and toes.
Why is apoptosis important in tissue homeostasis?	To maintain genetic integrity and cellular/tissue function.
Why is apoptosis important in neoplastic disease?	Because genes which control apoptotic mechanisms are compromised to promote tumour growth.
Describe the general process of apoptosis.	A 'death receptor' on the doom cell binds a signal molecule. Caspases are activated within the cell. The caspases destroy various intracellular components and the cell undulates into 'blebs' (cell fragments). A phagocyte attacks and engulfs the cell remnants and the cell components are degraded.
What are the two pathways that can result in apoptosis?	The EXTRINSIC pathway (in which signals from the external environment can signal the cell to apoptose - the death receptor pathway) and the INTRINSIC pathway (in which intracellular responses to damage such as radiation can cause apoptosis - the cell stress pathway).
Which receptors provide the major mechanism for the extracellular control of apoptosis?	DEATH
Describe the death receptors involved in the extrinsic apoptotic pathway.	They are part of the tumour necrosis factor receptor (TNFR) superfamily and contain a cytosolic death domain, e.g. the Fas-associated death domain (FADD), which is able to engage intracellular apoptotic machinery or mediate other cellular functions.
Describe the Fas death receptor.	The Fas death receptor is activated by a Fas ligand which mediates cell death: in peripheral deletion of mature T cells following an immune response, in killing virus-infected or cancer cells by cytotoxic T lymphocytes or natural killer cells, and in the killing of inflammatory cells.
The binding of the Fas ligand to the death domain of its transmembrane receptor activates which intracellular protein on the intracellular portion of the receptor?	FADD.
Once FADD is activated by Fas, what protein does its death effector domain recruit?	A precursor form of caspase-8.
Once the precursor of caspase-8 has been recruited by the death effector domain of FADD in the extrinsic apoptotic pathway, what protein self-cleaves to activate itself, which is also the last point in the apoptotic pathway that apoptosis can be stopped?	Caspase-8. This then goes on to activate downstream caspases such as caspase-9, resulting in apoptotic cell death. After caspase-8 is activated, the process is irreversible.
After caspase-8 is activated, describe the rest of the events in the extrinsic apoptotic pathway.	Activated caspase-8 may cleave tBID into BID which activates a member of the Bcl-2 family, BAK. This in turn results in release of cytochrome C from mitochondria. Cytochrome C promotes the formation of an apoptosome, a pin-wheel-like structure, which recruits SEVEN pro-caspases and activates them. For example, activated caspase-9 can activate caspase-3, demonstrating that this is a cascade. Substrates such as lamin, which composes the nuclear lamina, are broken cleaved and cellular structures are broken down. Caspase-8 -> tBID -> BID -> BAK -> cytochome C -> pro-caspases -> organelle breakdown.
What are caspases and what do they do?	Caspases are proteolytic cleaving enzymes which exist in their inactivate form in the cellular cytoplasm (or compartmentalised to avoid premature activation) and which can be activated autocatalytically or by other enzymes. Caspases directly disassemble cell structures by cleaving, reorganise and cleave the cytoskeleton, prevent DNA repair by cleaving important proteins, cut off contact with neighbouring cells, disintegrate the cell into apoptotic bodies for engulfment, induce signals for phagocytosis, and inactivate inhibitors such as Bcl-2.
What are the Bcl-2 proteins and what do they do?	The Bcl-2 proteins can be anti-apoptosis or pro-apoptosis. Bax and Bak are pro-apoptosis and induce cytochrome c release from mitochondria. Bcl-2 itself is anti-apoptosis, or apoptosis-inhibiting, and blocks Bax and Bak from promoting cytochrome c release. Conversely, a pro-apoptotic protein, BAD, inhibits Bcl-2 from doing this. The balance between the activities of the Bcl-2 proteins largely determines whether a mammalian cell lives or dies by apoptosis.
What are survival factors required for?	They are required by cells to prevent apoptosis and therefore grow and divide.
How do survival factors tend to act on cells?	By binding to cell membranes and causing anti-apoptosis Bcl-2 proteins to be transcribed and formed. Their absence results in intracellular programmed death being activated so in growing tissues, a limited supply may be released to competing cells to regulate tissue growth.
Give four examples of stimuli that can cause the intrinsic apoptotic pathway to be activated.	Radiation, toxins, free radicals, and absence of growth/survival factors.
Cellular damage results in the activation of which gene?	The p53 gene! ('The guardian of the genome')
Once activated, what does the p53 gene do?	It induces production/activation of Bax which both activates Bak and joins it in binding to mitochondria and initiating cytochrome c release. After this, the same caspase cascade is activated as in the extrinsic pathway and these caspases go on to cleave cellular components, along with activated pro-apoptotic Bcl-2 proteins.
What function do Bcl-2 proteins have in common with bacterial toxins?	The ability to form pores in organelle membranes (mitochondria, endoplasmic reticulum, nucleus), which can alter membrane permeability or potential or puncture it completely to disrupt its integrity.
List the events of the intrinsic apoptotic pathway (the 'cellular stress' pathway).	Cellular stress or survival factor absence activates p53 gene -> production/activation of Bax -> Activates Bak ->Bax and Bak induce mitochondria to release cytochrome c -> cytochrome c initiates apoptosome formation and further caspase activation -> activated caspase-9 activates caspase-3 -> caspases cleave substrates such as lamin of the nuclear lamina -> cellular structures are broken down.
Compare and contrast the extrinsic and intrinsic apoptotic pathways.	In both pathways, release of cytochrome c from mitochondria leads to a caspase cascade, but it is the stimulus which activates Bax and Bak which differs (p53 or caspase-8 and tBID).
What is 'looting' in apoptosis?	The apoptotic bodies produced by the caspases are phagocytosed into neighbouring cells as they contain intact mitochondria, ribosomes, lysosomes etc.
What happens to apoptotic bodies other than looting?	They are phagocytosed by macrophages signalled by caspases.
Why is the inflammatory response not initiated in apoptosis?	Because the cell contents is not allowed to leak into the extracellular space (due to phagocytosis either by neighbouring cells or macrophages).
Why do cells become small and rounded in apoptosis? What does this result in?	This shrinkage results from a loss of water and ions as cells become 'leakier'. It results in a loss of cell-cell adhesion with neighbouring cells.
What is 'blebbing' and why does it occur?	'Blebbing' or lobulation of the membrane tends to occur as it loses its structural integrity and these may go on to form the apoptotic bodies that are phagocytosed.
What are apoptotic bodies?	The cell bursts after significant intracellular degradation and the hydrophobic lipid bilayers quickly reform around cell contents to contain it (apoptotic bodies) for phagocytosis. These bodies can be seen inside macrophages up to 9 hours after phagocytosis.
Describe some of the intracellular events during apoptotis.	The cytoskeleton is broken up and collapses; the chromatin condenses to form 'half-moon' structures and the nucleus itself may break up into fragments; the DNA fragments form 'apoptotic ladders' when viewed on agarose gel under UV light.
Describe an oncogene that is involved in the regulation of apoptosis.	The Bcl-2 protein family can be regulated by the c-myc gene. These proteins comprise anti-apoptosis and pro-apoptosis and the mutation in the initiation/promotion of cancer is usually in favour of the anti-apoptosis proteins.
Describe a tumour suppressor gene involved in the regulation of apoptosis.	The product of the p53 gene activates Bax and the rest of the caspase cascade. Given that it initiates intracellular apoptosis, its loss in mutation in neoplastic growth means that a mutated cell is not recognised as abnormal and apoptosis is not initiated.
Why might one need to discriminate between apoptotic and necrotic cell death?	To see how a drug induces cell death.
What is the method for carrying out a cell viability test to differentiate between apoptosed/necrosed cells and living, healthy cells?	Add trypan blue and propidium iodide - these will only enter a non-viable, permeable ('leaky') cell.
What will be SEEN in apoptosed and necrosed cells in a cell viability test?	In both apoptotic and necrotic cells, the nuclei will be stained red by propidium iodide and the rest of the cell will be stained blue by trypan blue.
What are the benefits of using a cell viability test, rather than any other method, to determined whether cells are alive or necrotic/apoptotic?	It is a quick, easy, and inexpensive test.
What is the method for the terminal d-UTP 'nicked-end' labelling (TUNEL) assay for determining whether cells are alive or apoptotic/necrotic?	After DNA fragmentation, terminal ends of nucleic acids are exposed. An enzyme can be used to add nucleotides (dUTPs) which are labelled with a fluorescent marker which can then be viewed using fluorescence microscopy.
What will be SEEN in a terminal dUTP nicked-end labelling (TUNEL) assay to determine whether cells are alive or necrotic/apoptotic? Why might this test not be ideal?	In both apoptotic and necrotic cells, DNA fragments will fluoresce under appropriate light. However, cells that have undergone severe DNA damage may be marked too - so if testing the efficacy of a chemotherapeutic drug, could not see the difference between malignant and dead cells!
What is the mechanism behind the Apoptest™?	Phosphotidyl serine is a phospholipid usually present on the inner leaflet of the membrane. In early apoptosis, it is flipped outside. A fluorescent marker is attached to Annexin V and Annexin V binds to phosphotidyl serine.
What is seen when the Apoptest™ is used to detect apoptosis?	The dye will fluoresce under UV light. The percentage present can be indicative of the stage of apoptosis and be shown on a flocytometer as different colours.
What is seen if the Apoptest™ is used to detect necrosis?	Nothing - it is not used to detect necrosis as the flipping of phophotidyl serine does not occur consistently enough to test.
What is the method behind DNA fragmentation/laddering for testing for apoptotic/necrotic cells?	The DNA is isolated and run on agarose gel by electrophoresis. Fluorescent markers are used to visualise. The idea is that DNA is broken down neatly in apoptosis and not properly broken down at all in necrosis.
What is seen on the agarose gel in a DNA fragmentation/laddering test when apoptotic cells are present?	Clear distinct bands in a continuous spread /column down the gel - like a ladder.
What is seen on the agarose gel in a DNA fragmentation/laddering test when necrotic cells are present?	A smear of messily cut DNA, most of this stays at the top of the gel.
What is autophagy?	Autophagy is another form of programmed cell death, along with apoptosis. It tends to be seen in starving cells, and organelles are broken down and recycled for energy, similarly to fat and protein being broken down for energy in holistic starvation but on a cellular scale.
Autophagy uses lysosomes. Describe the contents of a lysosome.	Lysosomes contain around 40 hydrolytic enzymes including those which degrade proteins, nucleic acids, oligosaccharides, and phospholipids. These enzymes are kept in an acidic environment as they are optimally active at ~pH5 - so their escape in to the ~pH7.2 cytosol would involve limited damage.
How are organelles taken up into lysosomes in autophagy?	The organelle or structure is enclosed in a double membrane, creating an autophagosome, which then fuses with the lysosome for enzymatic breakdown. It is not known what marks an organelle for such destruction.
Why can autophagy be seen primarily as a survival mechanism rather than a death mechanism?	In autophagy, the cell 'hopes' that favourable conditions will be restored. It may not result in death, which is why it can be seen as a survival mechanism.
What is the name of the 'structure' formed when sections of double membrane begin to form around organelles marked for autophagy?	A phagophore.
What protein links the double membrane from a phagophore together to form a more established autophagosome?	There is recruitment of LC3B.
What is the name of the structure formed when a lysosome fuses with an autophagosome?	An autolysosome.
What happens to the LC3B proteins which link the double membrane of the autophagosome together when the autophagosome has fused with a lysosome to form an autolysosome?	The LC3B proteins dissociate from the structure so that only the lysosome is left.
What are the products of autophagy?	Amino acids, fatty acids, and other metabolic monomers.
In which programmed cell death process are phagocytes involved?	Apoptosis.
In which programmed cell death process(es) is there cell membrane 'blebbing'?	In both apoptosis and autophagy, though in autophagy it is used to form the autophagosome.
In which programmed cell death process(es) is DNA laddering seen?	Only in apoptosis as DNA is not broken down in autophagy.
In which programmed cell death process(es) are organelles preserved in vacuoles?	In apoptosis only, to be passed to other cells ('looting'), as the purpose of autophagy is to break down the organelle to provide energy for the cell.
In which programmed cell death process(es) is the cytoskeleton preserved?	In autophagy only, as the cell may survive.
In which programmed cell death process(es) are caspases involved?	Apoptosis.
Why is apoptosis a target for treatment and prevention of cancer?	Because drugs which encourage apoptosis tend to result in 'cleaner' cell death and are a safer treatment than obliterating cancerous cells with radiation, for example.
What property, other than being safe, do apoptosis targets in cancer treatment/prevention have?	They are more specific than treatments such as radiation.
Why are some cancers reduced in people who habitually take aspirin?	Because NSAIDs such as aspirin promote apoptosis and so encourage 'cleaner' cell death.
Why might butyrate be able to be used to target gastrointestinal neoplasms?	Because it encourages gastrointestinal proliferation and apoptosis and therefore promotes 'cleaner' cell death.
What is necrosis?	Necrosis is a form of cell death that is not pre-programmed but is a result of physical trauma, toxins or ischaemia.
What are the initial, reversible cell changes in necrosis?	Mitochondrial swelling and cell blebbing.
If a noxious stimulus persists beyond the reversible changes in necrosis, how does necrosis persist and cause inflammation?	Necrotic cells swell, their organelles deteriorate, and finally the cells rupture. The cell contents released this way include digestive enzymes that damage adjacent cells and trigger an inflammatory response.
List the seven types of necrosis.	Coagulative, liquefactive, caseous, fat, fibrinoid, gangrenous, and autolytic.
What are the two overall categories of damage that can cause necrosis?	Membrane damage and mitochondrial damage.
What can cause membrane damage to the point of necrosis?	Free-radicals, an immune-mediated response, and bacterial toxins.
What can cause mitochondrial damage to the point of necrosis?	Low oxygen and glucose (from ischaemia), and poisons such as cyanide which disrupt the electron transport along the mitochondrial membrane.
What is pyknosis?	Cell shrinkage - transcription is halted and there is loss of cytoplasmic RNA
What is karyorrhexis?	Messy DNA fragmentation - nuclease fragmentation of the nucleus.
What is karyolysis?	The complete dissolution of the nucleus, along with partly denatured proteins
How do cells appear histologically when undergoing pyknosis?	There is eosinphilia of the cytoplasm (stains pink) and the basophilic nucleus appears small.
How do cells appear histologically in karyorrhexis?	There is nuclear fragmentation, the cells remains small and eosinophilic, and membrane irregularities can be seen (the beginning of 'blebbing').
How do cells appear histologically in karyolysis?	There is no discernible nucleus, the eosinophilic cytoplasm remains, and there is a marked contrast to other cells.
What is coagulative necrosis?	Coagulative necrosis is a form of cell death that is usually a result of tissue hypoxia, such as that following a myocardial infarction. It tends to occur in solid organs, such as the heart or kidneys, and, grossly, the dead tissue appears pale and firm, as if cooked.
How long might coagulative necrosis persist?	The process of pyknosis and karyorrhexis/karyolysis might continue for weeks, but in particularly large areas of necrosis, such as that caused by occlusion of a coronary artery, the central region may be inaccessible to the inflammatory response and the necrotic tissue can persist, perhaps even for years.
What is the 'snapshot' situation in coagulative necrosis?	This is a temporary situation, before the necrotic cells have been removed by phagocytes, in which the cell outline and architecture may be intact despite the cells being dead.
How does liquefactive necrosis predominately differentiate from coagulative necrosis?	It differs in that enzymatic digestive processes predominate over denaturing processes. The result of the powerful hydrolytic enzymes is semi-liquid dead tissue. The enzymes degrade both intracellular and extracellular components to produce a 'proteinaceous soup'.
What are the two circumstances in which liquefactive necrosis develops?	In the sharply localised response of polymorphonuclear leukocytes to a bacterial infection, or as a result of tissue hypoxia following cerebral artery occlusion and infarction.
How might the immune response to a bacterial infection result in liquefactive necrosis?	The bacterial infection can result in a sharply localised collection of the polymorphonuclear leukocytes of the acute inflammatory response. These are armed with potent hydrolases which produce that rapid death and dissolution of the tissue.
How might cerebral artery occlusion result in liquefactive necrosis?	Cerebral artery occlusion and infarction as a result of ischaemia/hypoxia often results in a liquefactive necrotic cavity being produced that cannot be attributed to an acute inflammatory response (as it is in the brain). It is thought to be a result of cells of the central nervous system containing more or different hydrolases.
How long might the liquefactive necrotic cavity of fluid and necrotic debris (as a result of cerebral artery occlusion) persist?	It can persist for the life of a person.
What may necrosis following ischaemia also be a result of (other than damaged central nervous system cells leaking hydrolases)?	It may be a result of damaged mitochondria as even a reperfusion injury (such as influx of Ca2+ to the mitochondria) may cause damage.
What is caseous necrosis typically caused by (and what is it classically a lesion of)?	Caseous necrosis is typically caused by mycobacteria or foreign substances (e.g. asbestos). It is characteristic of the tuberculous granulomas seen in tuberculosis.
Grossly, how does the tissue appear in caseous necrosis?	The tissue is soft and white and resembles crumbly cheese.
Why can caseous necrosis be considered a cross between liquefactive and cogulative necroses?	Because the necrotic area is not quite liquid but the cells do not retain their outlines.
What is the appearance of the dead cells that persist indefinitely in caseous necrosis?	Amorphous, coarsely granular, and eosinophilic debris.
What is present in the centre of a tuberculous granuloma?	The accumulated mononuclear cells, which are mediating the chronic inflammatory response to the offending bacterium, are killed as a result of the potent effect of the waxes of the mycobacterial cell walls. This forms pus - a collection of dead immune cells.
In what tissue does fat necrosis occur (only)?	Adipose tissue! Such as that of the breast.
What does fat necrosis typically result from?	Pancreatitis or trauma.
Why can fat necrosis follow pancreatitis or pancreatic trauma?	Because lipase is released, either into the peripancreatic tissues or the peritoneal cavity or into the blood so that they spread. The acinar cells of the pancreas are damaged and phospholipases and proteases attack the plasma membranes of the cells while lipase hydrolyses the triglycerides that are released into fatty acids. The fatty acids are precipitated as calcium salts.
Why is necrotic fat recognised early?	Due to its greyish discolouration.
What forms the palpable lump in fat necrosis?	The combination of necrotic fat with calcium causes a gritty, chalky white and irregular area to precipitate in otherwise normal adipose tissue. This creates a lump.
Where does fibrinoid necrosis most commonly occur?	In arteries.
What does the term 'fibrinoid necrosis' describe?	The most common appearance in cases of vasculitis and hypertension.
What is deposited in the damaged blood vessels in fibrinoid necrosis? What classes this injury as necrotic?	Fibrin is deposited in the damaged blood vessels. The cells have indistinct structures (in the walls of the vessel) and stain intensely with eosin - it is this eosinophilia caused by increased binding of the cytoplasmic proteins that classes this injury as necrotic.
What does the word 'gangrene' describe?	Black, dead tissue.
If liquefactive necrosis can be called wet gangrene, what can gangrene be called?	Dry liquefactive necrosis.
Where is gangrene most commonly seen?	In the lower limbs of patients with severe atherosclerosis as there is restricted peripheral blood supply which results in ischaemia.
How does one characterise dry and wet gangrene?	If the pattern of necrosis mostly follows coagulative necrosis, it is dry gangrene. If infection with Gram-negative bacteria converts it to liquefactive necrosis, it is wet gangrene.
Where is autolytic change/autolytic necrosis most commonly seen?	This is most commonly seen in post mortem tissues when the heart has stopped pumping and all the tissues become irreversibly ischaemic.
In autolytic necrosis, enzymes leak from the cells and digest adjacent structures. Why is there no transport or inflammatory response?	Because there is no circulation and the tissue is dead.
What is a characteristic cellular feature in coagulative necrosis?	The cellular outlines remain.
What is a characteristic cellular feature of liquefactive necrosis?	There is loss of cellular detail.
What is a characteristic cellular feature of caseous necrosis?	Loss of cellular detail and calcification.
What is a characteristic feature of fat necrosis?	It occurs in fatty tissues and there is calcification.
What is a characteristic cellular feature of fibrinoid necrosis?	Markedly eosinophilic vessel walls.
Cells tend to keep Ca2+ out when alive so once they have been killed they lose this ability and there is a large influx of calcium. What does this combine with once in the cell?	The Ca2+ combines with phosphates within the mitochondria to produce hydroxyapatite crystals. The large number of mitochondria mean that a cell becomes calcified, as well as other intracellular components that Ca2+ may react with.
Why might there be extracellular calcium deposition in necrotic tissues? Why might this be dangerous?	Because degenerating cells produce vesicles containing Ca2+. The danger is dependent on where the deposit forms - if it forms on the heart valves, for instance, it might affect the function.
What distance must a tumour cell be within from a blood supply in order to avoid autophagy/necrosis?	~85µm
Why can necrosis be an early indicator of cancer?	Because ischaemia in some areas causes cancer cells to die and necrose as extra angiogenesis is restricted - perhaps by vascular endothelial growth factor (VEGF).
What is Avastin and how is it used in cancer treatment?	Avastin is a drug that mimics vascular endothelial growth factor (VEGF) in its ability to restrict angiogenesis and therefore neoplastic growth.
How might one detect calcification in necrosis?	USing X-ray - this allows it to be detected early.
How does the inflammation from necrosis promote survival in neoplastic cells?	The cells of the inflammatory response, including macrophages and neutrophils, release cytokines and other cell mediators to signal and proliferate each other but this can also affect the neoplastic cells and promote their survival.
Which form of cell death requires energy from the cell?	Apoptosis requires energy from the targeted cell.
Which process of cell death usually generates an inflammatory reaction?	Necrosis because the cell contents are not contained within membranes so the enzymes can damage/break down surrounding cells.
Which form of cell death happens to groups of cells rather than individual cells?	Necrosis.
In which form of cell death is there cell swelling/shrinkage? Why?	In necrosis there is cell swelling due to influx of water as the cell begins to lose structural integrity. In apoptosis there is cell shrinkage as the cytoskeleton is disassembled and fails to continue to support the membrane.
What is the overall cause for apoptosis? And for necrosis?	The overall cause for apoptosis can be physiological, dvelopmental or pathological, whereas for necrosis it is only pathological.

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Apoptosis, autophagy and necrosis

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