Mechanisms of Disease II - Cell Death and Cell Damage

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

Question 2

What causes necrosis

Answer 2

Usually lack of blood supply as a result of:

• injury
• infection
• cancer
• infarction
• inflammation

Question 3

Describe the step-by-step process of necrosis

Answer 3

1. Result of an injurious agent or event.
2. Initial events are reversible, later ones are not.
3. Lack of oxygen prevents ATP production.
4. Cells swell due to influx of water (ATP is required for ion pumps to work).
5. Lysosomes rupture; enzymes degrade other organelles and nuclear material hapzardly
6. Cellular debris released, triggering inflammation

Question 4

What are the nuclear changes in necrosis

Answer 4

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

Question 5

What are the cytoplasmic changes in necrosis

Answer 5

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

Question 6

What are the biochemical changes in necrosis

Answer 6

1. Release of enzymes such as creatine kinase or lactate dehydrogenase
2. Release of other proteins such as myoglobin

These biochemical changes are useful in the clinic to measure the extent of tissue damage!

Question 7

What is Apoptosis and what are its functions

Answer 7

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

Involved in:

• Embryogenesis
• Metamorphosis
• Normal tissue turnover
• Endocrine-dependent tissue atrophy
• A variety of pathological conditions

Question 8

Describe the step-by-step process of Apoptosis

Answer 8

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.
5. New molecules are expressed on vesicle membranes that stimulate phagocytosis without an inflammatory response.

Question 9

What are the cytoplasmic changes of Apoptosis

Answer 9

1. Shrinkage of cell. Organelles packaged into membrane vesicles.
2. Cell fragmentation. Membrane bound vesicles bud off.
3. Phagocytosis of cell fragments by macrophage and adjacent cell.
4. No leakage of cytosolic components.

Question 10

What are the nuclear changes in Apoptosis

Answer 10

1. Nuclear chromatin condenses on nuclear membrane.
2. DNA cleavage.

Question 11

What are the biochemical changes in Apoptosis

Answer 11

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 12

Describe how normal, apoptic and necrotic DNA would look like in DNA fragmentation

Answer 12

Normal - No banding
Apoptic - distinct bands visible
Necrotic - Smear like banding pattern

Question 13

What are some examples of Apoptosis

Answer 13

Metamorphosis - Tadpole’s tail is lost

Interdigital web loss in mouse paw development

Question 14

What are the two types of apoptosis

Answer 14

Intrinsic:
DNA damage – p53-dependent pathway
Interruption of the cell cycle
Inhibition of protein synthesis
Viral Infection
Change in redox state

Extrinsic:
Withdrawal of survival factors e.g. mitogens
Extracellular signals (e.g. TNF)
T cell or NK (Natural Killer) (e.g. Granzyme).

Question 15

What are caspases

Answer 15

Caspases are cysteine protease
Caspases are the point of convergence for causes of apoptosis.
They form an activation cascade, where one cleaves and activates the next (analogous to kinase cascades)

Question 16

Describe caspase activation

Answer 16

An inactive procaspase Y is cleaved at 2 sites by an active cascade X.
this yields a prodomain and an active caspase Y

Question 17

Describe the caspase cascade

Answer 17

Initiator caspases (8&9) few molecules, cleave processes into their active form.
They then continue cleaving more procaspases to form a cascade. The newly formed caspases are called effectors

Question 18

What is the effect of caspase activation

Answer 18

Caspase activation leads to characteristic morphological changes, such as shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebbing.

Question 19

How is the initiator caspase activated

Answer 19

Initiator caspases activate themselves when in close proximity
Activation, therefore, means bringing initiator caspases together

Question 20

How is the extrinsic apoptosis pathway activated

Answer 20

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

Question 21

Explain the Extrinsic Apoptosis pathway

Answer 21

When an extrinsic factor binds to its receptor, it brings death domains closer together and creates an environment for death adapters to join them. This leads to an increase in death effectors, which allows procaspase-8 to join them as well. The proximity of these components enables them to undergo auto-proteolysis in that environment, which activates the caspase.

Question 22

How is the intrinsic pathway activated

Answer 22

Induced by cytochrome c released from mitochondria

Note: growth factor withdrawl (extrinsic apoptosis) an exception that uses cytochrome c

Question 23

What is cytochrome c

Answer 23

Mitochondrial matrix protein
Known for many years to be released in response to oxidative stress by a “permeability transition”
Any inducers of the permeability transition also eventually induce apoptosis.

Question 24

Explain the intrinsic Apoptosis pathway

Answer 24

Cytochrome C binds its receptor which brings together caspase recruitment domains together. This causes procaspase 9 to oligomerise with the domain and is brought near each other. They undergo auto-proteolysis which activates it.

Question 25

How is the release of cytochrome c regulated from the mitochondria

Answer 25

A family of proteins called the BCL-2 family form a pore on the mitochondrial membrane which allows cytochrome c to leave via it.
These proteins can either be pro or anti apoptotic.

Question 26

Explain how the Bax protein works

Answer 26

Bax proteins form a pore which allows cytochrome c to pass through. this is pro-apoptotic

Question 27

Explain how the Bax + BCL-2 protein works

Answer 27

BCL-2 binds to the spore opening and stops cytochrome c from leaving.
Anti-apoptic

Question 28

Explain how the Bax + BCL-2 + BAD protein works

Answer 28

BAD binds to BCL-2 preventing BCL-2 to bind to the pore.
Pro-apoptic

Question 29

What regulates the BCL-2 family proteins

Answer 29

Transcription factors and growth factors

Question 30

How does TP53 cause apoptosis

Answer 30

Transcription driven by TP53 causes more pores to be inserted into the membrane.
Causes more cytochrome c to leave causing apoptosis

Question 31

How do Growth factors regulate BCL-2 family proteins

Answer 31

Growth factors cause phosphorylation of BAD which stops it from binding BCL-2 which means the pores are blocked so no cytochrome c release.