Mechanisms of Disease II: Cell Death and Cell Damage

Question 1

What is the function of necrosis? What can happen if necrosis fails?

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 are the causes of necrosis?

Answer 2

Usually lack of blood supply, e.g.

- Injury such as a compression injury
- Infection most obvious example is gangrene
- cancer, 
- infarction, 
- Inflammation

Question 3

What are the steps to necrosis? (6)

Answer 3

Necrosis step-by-step
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.
4. Cells swell due to influx of water (because ATP is required for ion pumps to work).
5. Lysosomes rupture; enzymes degrade other organelles and nuclear material haphazardly
6. Cellular debris released, triggering inflammation

Question 4

What are the nuclear changes in appearance that occur during necrosis?

Answer 4

Nuclear Changes:

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

Question 5

What are the cytoplasmic changes in appearance that occur during necrosis?

Answer 5

Cytoplasmic changes

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

Question 6

What are the biochemical changes that occur during necrosis?

Answer 6

Biochemical changes:
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 are the functions of apoptosis?

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

What are the steps involved in apoptosis? (5)

Answer 8

1. Programmed cell death of one or a few cells (unlike necrosis)
2. Events are irreversible and energy (ATP) dependent. (unlike necrosis)
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 is the cytoplasmic microscopic appearance of a cell during apoptosis?

Answer 9

Cytoplasmic Changes
1. Shrinkage of cell. Organelles are 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 and biochemical changes that occur during apoptosis?

Answer 10

1. Nuclear chromatin condenses on nuclear membrane.
2. DNA cleavage.
   Biochemical changes:
3. Expression of charged sugar molecules on outer surface of cell membranes (recognised by macrophages to enhance phagocytosis)
4. Protein cleavage by proteases, caspases

Question 11

What are some examples of apoptosis? (8)

Answer 11

Cell death in embryonic hand to form individual fingers.

2. Apoptosis induced by growth factor deprivation (neuronal death from lack of NGF).
3. DNA damage-mediated apoptosis. If DNA is damaged due to radiation or chemo therapeutic agents, p53 (tumour suppressor gene product) accumulates. This arrests the cell cycle enabling the cell to repair the damage. If repair process fails, p53 triggers apoptosis.
4. Cell death in tumours causing regression.
5. Cell death in viral diseases (ie viral hepatitis).
6. Cell death induced by cytotoxic T cells (ie. Cellular immune rejection or vs. host disease).
7. Death of neutrophils during an acute inflammatory response.
8. Death of immune cells( both T and B lymphocytes) after depletion of cytokines as well of death of autoreactive T cells in the developing thymus

Question 12

What are the two types of apoptosis?

Answer 12

Intrinsic:
- DNA damage – p53-dependent pathway
- Interruption of the cell cycle
- Inhibition of protein synthesis
- Viral Infection- i.e. once virus is in the cell
- Change in redox state
Extrinsic- Relative to the cell not the body:
- Withdrawal of survival factors e.g. mitogens
- Extracellular signals (e.g. TNF)
- T cell or NK (Natural Killer) (e.g. Granzyme).

Question 13

What are caspases?

Answer 13

Caspases are the point of convergence for causes of apoptosis, e.g.
Caspases are cysteine proteases (cysteine aspartate-specific proteases)
Caspases form an activation cascade, where one cleaves and activates the next (analogous to kinase cascades)

Question 14

How are caspases activated?

Answer 14

Inactive caspase is called procaspase
It is cleaved in two places by active caspase X releasing this prodomain
The small and large subunit rearrange into its active configuration
Resulting in an active molecules of caspase Y

Question 15

What is the caspase cascade?*

Answer 15

It’s a cascade of caspases
But the downstream caspases also have other substrates that are not caspases
So you will get cleavage of cytosolic proteins and cleavage of the nucleus lamina proteins
The ones at the top are initiator caspases and the downstream ones with other substrates are effector caspases

Question 16

What is the effect of caspase activation?

Answer 16

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

Question 17

How do we activate the initiator caspase?

Answer 17

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

Question 18

What is extrinsic apoptosis induced by?

Answer 18

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

Question 19

Who is involved in ligand-induced multimerisation?

Answer 19

A ligand which will bind to the transmembrane receptor on the cell membrane
Importantly for these extrinsic types of apoptosis we have these intermediate proteins called death adaptor proteins
We have our caspase in its inactive procaspase form as procaspase-8 in humans
The receptor and death adaptor proteins have death domains
The death adaptor proteins and procaspase have death effector domains
These domains are so they can dimerise or multimerise

Question 20

How is TNF an example of ligand-induced multimerisation?

Answer 20

Tumour necrosis factor is bound to the ligand binding domain of its receptor and its brought together these death domains which creates an environment where oligomerisation with other proteins is favoured in this case is FADD
This has then created an environment with a lot of death effector domain proteins and those death effector domains in turn recruit procaspase 8
This has formed a death-inducing signalling complex (DISC)
The procaspase-8 proteins in close proximity causes autoproteolysis which causes activation of the caspases

Question 21

What is intrinsic apoptosis induced by?

Answer 21

Induced by cytochrome c released from mitochondria

Note: growth factor withdrawal (extrinsic apoptosis) an exception that uses cytochrome c… (see later)

Question 22

What is cytochrome C?

Answer 22

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 23

What is involved in cytochrome C-induced apoptosis?

Answer 23

In this case instead of a death adaptor protein there is an APAF-1 protein (apoptotic protease activating factor)
It has a cytochrome C binding site, an APAF domain and a caspase recruitment domain (CARD)
The initiator caspase for intrinsic apoptosis is procaspase-9

Question 24

What happens in cytochrome C-induced apoptosis?

Answer 24

The same thing happens as in extrinsic apoptosis

This whole complex is referred to as an apoptosome

Question 25

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

Answer 25

BCL-2 is the name of the family but its also a member of the family
A pore made of BCL-2 family proteins through which cytochrome C can be release

Question 26

How can BCL-2 proteins be pro- and anti-apoptotic? Give an example

Answer 26

Anti-apoptotic- bcl-2, bcl-XL, others repress cytochrome c release
Pro-apoptotic- Bax, Bad, Bid, others facilitate cytochrome c release
Some are not membrane proteins, some are cytoplasmic predominantly
All have a BH3 domain used to form dimers

Example; Bax and bcl-2
BAX creates a pore so cytochrome C is release
When BCL-2 is also expressed it binds to BAX and blocks the pore
When BAD is also added that binds very strongly to BCL-2 so it displaces all the BCL-2 form the BAX pore

Question 27

If BCL-2 family proteins regulate cytochrome C release from mitochondria, what regulates BCL-2 proteins?

Give an example?

Answer 27

Transcription example; TP53
DNA damage triggers TP53 and the expression of TP53 target genes, a key one being BAX
Lots more BAX protein inserts into the membrane and ultimately new pores that cannot be blocked because there is not enough BCL-2 such that now cytochrome C is release and death ensues
Not the only mechanism but an essential one

Phosphorylation example; growth factors
The growth factors acting as survival signals will activate a serine threonine kinase known as Akt or PKB
This kinase can phosphorylate BAD which becomes sequestered in the cytoplasm because of this it is no longer available to bind to BCL-2 allowing BCL-2 to block the pores
When growth factors are removed there is nothing to inhibit BAD so it can bind to BCL-2 which allows cytochrome C to be released and death ensues