Mechanisms of Disease II

Question 1

What is the function of necrosis?

Answer 1

Removes damaged cells from an organism. Failure to do so may lead to chronic inflammation. Necrosis causes acute inflammation to clear cell debris via phagocytosis in order to prevent a more damaging chronic inflammatory situation.

Question 2

What are the causes of necrosis?

Answer 2

Usually lack of blood supply e.g., injury, infection, cancer, infarction, inflammation.

Question 3

Explain how necrosis takes place.

Answer 3

1. Result of an injurious agent or event – whole groups of cells are affected.
2. Initial events are reversible, later ones are not.
3. Lack of oxygen prevents ATP production (as normal glycolysis, TCA etc. cannot work).
4. Cells swell due to influx of water (ATP is required for ion pumps to work) – from osmosis. This changes osmomolarity of cytoplasm and puts more pressure on membranes within the cell due to expanded volume.
5. Lysosomes rupture; these release enzymes which degrade other organelles and nuclear material haphazardly and random manner.
6. Swelling continues, membrane expands too much such that cellular debris released, triggering inflammation – principally phagocytosis to remove the debris.

Question 4

When necrosis is still at a reversible stage, what is happening in a cell?

Answer 4

ATP production has reduced such that the ATP-dependent ion pumps are no longer able to maintain correct osmotic balance so water is entering the cell. The cell undergoes changes e.g., mitochondria larger and expanded, changes in pattern of chromatin and other damage to organelles. Still reversible as cell can return to normal if oxygenated again. Pumps would be able to address increased osmomolarity and return osmotic balance to normal.

Question 5

What are the nuclear changes when a cell undergoes necrosis?

Answer 5

1. Chromatin condensation/shrinkage.
2. Fragmentation of nucleus.
3. Dissolution of the chromatin by DNAse.

Question 6

What are the cytoplasmic changes when a cell undergoes necrosis?

Answer 6

1. Opacification: (transparent to solid white (like egg white)) protein denaturation & aggregation.
2. Complete digestion of cells by enzymes causing cell to liquify (liquefactive necrosis

Question 7

What are the biochemical changes when a cell undergoes necrosis?

Answer 7

1. Release of enzymes such as creatine kinase or lactate dehydrogenase. These enzymes should only be within the cell not extracellular environment!
2. Release of other proteins such as myoglobin (into bloodstream) when muscle injured.

Question 8

What are the functions of apoptosis?

Answer 8

Selective process for the deletion of superfluous, infected or transformed cells (cancerous).

Question 9

What is apoptosis involved in?

Answer 9

Embryogenesis, metamorphosis, normal tissue turnover, endocrine-dependent tissue atrophy (extracellular signalling telling the cell to induce apoptosis) and a variety of pathological conditions.

Question 10

Explain how apoptosis takes place.

Answer 10

1. Programmed cell death of one or a few cells.
2. Events are irreversible and energy (ATP) dependent.
3. Cells shrink as the cytoskeleton is disassembled.
4. Orderly packaging of organelles and nuclear fragments into membrane bound vesicles.

Question 11

How is apoptosis different to necrosis?

Answer 11

1. Programmed cell death of one or a few cells. Necrosis is whole groups of cells.
2. Apoptosis – all events are irreversible whereas necrosis early stages are reversible.
3. Apoptosis – energy (ATP) dependent whereas apoptosis is not dependent.
4. Apoptosis – cell contents packaged in vesicles in an orderly manner whereas necrosis just ruptures into extracellular environment.
5. New molecules are expressed on vesicle membranes that stimulate phagocytosis without an inflammatory response. Quite a clean disposal.

Question 12

What are the cytoplasmic changes when a cell undergoes apoptosis?

Answer 12

1. Shrinkage of cell. Organelles packaged into membrane vesicles.
2. Cell fragmentation. Membrane bound vesicles to bud off.
3. Phagocytosis of cell fragments by macrophages and adjacent cells.
4. No/very minimal leakage of cytosolic components into extracellular environment which could cause inflammation. Sometimes there is some inflammation which causes secondary necrosis.

Question 13

What are the nuclear changes when a cell undergoes apoptosis?

Answer 13

1. Nuclear chromatin condenses onto the nuclear membrane.

2. DNA cleavage.

Question 14

What are the biochemical changes when a cell undergoes apoptosis?

Answer 14

1. Expression of charged sugar molecules on outer surface of cell membranes (recognised by macrophages to enhance phagocytosis).
2. Protein cleavage by proteases - caspases.

Question 15

What can you see in DNA fragmentation?

Answer 15

Characteristic laddering of chromosomal DNA caused by apoptosis. A ‘smear’ is produced for necrotic cells.

Question 16

Give some examples of apoptosis.

Answer 16

Metamorphosis – tadpole’s tail lost by apoptosis - and interdigital web loss – mouse paw development.

Question 17

Name some signals that cause cell survival.

Answer 17

Growth factors, cytokines and cell-cell and/or cell-matrix contacts.

Question 18

Name some signals that cause cell apoptosis.

Answer 18

Disruption of cell-cell and/or cell-matrix contacts, lack of growth factors, death domain ligands and DNA damaging agents.

Question 19

What are caspases?

Answer 19

Caspases are the point of convergence for causes of apoptosis e.g., extrinsic and intrinsic causes both use caspases to cause apoptosis. They are cysteine proteases (cysteine aspartate-specific proteases). They form an activation cascade, where each caspase sequentially cleaves and activates the next (analogous to kinase cascades).

Question 20

How are caspases activated?

Answer 20

Inactive procaspase Y has three parts to protein. It’s cleaved by active caspase X in two places. This causes the release of the end terminal prodomain. This results in an active molecule of active caspase Y.

Question 21

Explain the caspase cascade.

Answer 21

Initiator caspase (e.g., 8, 9) activate downstream caspases which, in turn, will activate even more further downstream. These are called effector caspases (e.g., 1,3,6,7) which have an additional substrate. They don’t just activate caspases, but also cleave cytosolic proteins and nuclear lamins (due to their substrate e.g., substrate lamin A and B causes loss of nuclear envelope function). Furthermore, this is a signal amplification cascade as it is not linear but causes rapid increase of caspases.

Question 22

What does caspase activation cause?

Answer 22

Characteristic morphological changes, such as shrinkage (degradation of microtubule cytoskeleton), chromatin condensation, DNA fragmentation and plasma membrane blebbing.

Question 23

How is the initiator caspase activated?

Answer 23

Initiator caspases activate themselves when in close proximity hence activation, therefore, means bringing initiator caspases together.

Question 24

What causes extrinsic apoptosis?

Answer 24

Induced by ligand binding to receptors, causing receptor dimer- (or multimer-) isation.

Question 25

What are the key players in extrinsic apoptosis?

Answer 25

Extracellular ligand binds to its transmembrane receptor on the cell membrane. The receptor is made up of an extracellular ligand binding domain and intracellular death domain. There are also intermediate proteins called the death adaptor proteins. They are made up of a death domain and death effector domain. There is also the inactivated initiator caspase called procaspase -8. This is made up of the death effector domain and protease domain. Shared domains allow the proteins to bind together.

Question 26

How does extrinsic apoptosis take place?

Answer 26

Through death-inducing signalling complex (DISC).

Question 27

What causes intrinsic apoptosis?

Answer 27

Induced by cytochrome C released from mitochondria.

Question 28

What is cytochrome C?

Answer 28

A mitochondrial matrix protein. It is released in response to oxidative stress by a ‘permeability transition.’

Question 29

What are the key players in cytochrome c-induced apoptosis?

Answer 29

APAF-1 (apoptotic protease activating factor) is made up of cytochrome c binding site, APAF domain and CARD (caspase recruitment domain). Initiator procaspase-9 is made up of CARD and protease domain.

Question 30

How does intrinsic apoptosis take place?

Answer 30

Cytochrome c is released from mitochondria. It then binds to the binding domain on APAF-1. This brings the APAF-1 molecules together and multimerization occurs. Recruitment of procaspase-9 causes autoproteolysis due to close proximity of protease domains. This is called an apoptosome.

Question 31

How is the release of cytochrome c from the mitochondria regulated?

Answer 31

A pore made of BCL-2 family proteins. This pore allows release of cytochrome c from inner to outer mitochondrial membrane. However, not all of them are membrane proteins and some are cytoplasmic predominantly. But they all have a BH3 domain used to form dimers, trimers etc.

Question 32

Describe the two types of BCL-2 proteins.

Answer 32

1. Anti-apoptotic: bcl-2, bcl-XL, others. These repress cytochrome c release allowing cells to remain alive.
2. Pro-apoptotic: Bax, Bad, Bid, others. These facilitate cytochrome c release causing cell death.

Question 33

If bcl-2 family proteins regulate cytochrome c release from mitochondria, what regulates bcl-2 proteins?

Answer 33

It is mainly due to gene expression and post-translational modification. This can be fully answered through an example. In a cell that is not apoptotic, there is a pore present with bcl-2 proteins. But with DNA damage, TP53 is activated and this causes expression of TP53 target genes. One of these is BAX so as expression increases, it inserts itself into the membrane and makes new pores that cannot be blocked by bcl-2. This allows release of cytochrome c and therefore, cell death.