Mechanisms of Disease 2 :Cell Damage and Cell Death

Question 1

What is the function of necrosis?

Answer 1

• Removes damaged cells from an organism so that they do not cause chronic inflammation (but necrosis causes acute inflammation)

Question 2

Why does necrosis cause inflammation?

Answer 2

-Necrosis induces its own acute inflammation to clear cell debris via phagocytosis to avoid more chronic inflammation

Question 3

What can cause necrosis?

Answer 3

Mainly due to a lack of blood supply
This can result because of :
-Injury
-Infection
-Cancer
-Infarction
-Inflammation

Question 4

Why does a lack of blood supply cause necrosis ?

Answer 4

Tissue moves away from blood vessel = ⬇ partial pressure of oxygen.
+ ⬇ pH as we move away from blood vessel

Question 5

What is the step by step process of necrosis ?

Answer 5

1. Injury
2. ⬇ oxygen = no ATP production
3. ATP is required for ion channels on cell membrane, so ATP ion pumps stop working. Osmosis causes influx of water. Cell will swell.
4. Osmolality = lysosomes rupture, releasing enzymes which degrade organelles + nuclear material.
5. Cell membrane ruptures = cellular debris released = triggers inflammation

Question 6

What is the difference between apoptosis and necrosis?

Answer 6

Necrosis -Whole group of cells are affected
Apoptosis-Certain cells only
Necrosis-Triggered by external factors
Apoptosis -Cell death generated by normal healthy processes
Necrosis initial events are reversible however apoptosis is not

Question 7

At what stage is necrosis reversible?

Answer 7

It is reversible where not many organelles have expanded and not too much damage meaning if the cell is re-oxygenated , resupply ATP and the swelling can be decreased.

Question 8

Outline the necrosis microscopic appearance

Answer 8

1.Chromatin condenses and shrinks
2There is fragmentation of the nucleus, chromosomal DNA is in pieces
3.Dissolution of the chromatin by DNases

Question 9

Outline the cytoplasmic changes during necrosis

Answer 9

• Opacification-Protein denaturation and aggregation (not clear anymore becomes white)
• Complete digestion of cell by enzymes causing cell to liquify (liquefactive necrosis(Lose cell structures)

Question 10

Outline the biochemical changes during necrosis

Answer 10

Enzyme release e.g. creatine kinase /lactate dehydrogenase

Release of proteins e.g. myoglobin

This can be used to measure tissue damage extent

Question 11

Outline Astrocytoma

Answer 11

Tumour of astrocytes that form the supportive tissue of the brain.

Middle of tumour mass is necrotic because as tumour grows, vascularisation becomes further from the middle.

Cancer will try to make its own vascularisation = angiogenesis

Question 12

Apoptosis =

Answer 12

Apoptosis selectively removes unnecessary/infected/transformed cells (cancerous)

Question 13

What is apoptosis involved in?

Answer 13

Embryogenesis 
Metamorphosis
Normal tissue turnover (life span)
Endocrine-dependent tissue atrophy (extracellular ligands)
Pathological conditions

Question 14

Describe what Apoptosis is

Answer 14

Apoptosis is the programmed cell death of 1/few cells.

Irreversible + requires ATP(energy).

Question 15

What is the Apoptosis step by step

Answer 15

1. Cell shrinks due to cytoskeleton disassembly.
2. Cell contents are packaged into membrane-bound vesicles.
3. Vesicles express molecules that stimulate phagocytosis without causing an inflammatory response.

Question 16

Outline the apoptosis microscopic appearance

Answer 16

1. Cell shrinks + organelles are packaged into vesicles.
2. Cell fragmentation, membrane-bound vesicles will bud off.
3. The cell fragments are phagocytosed by macrophages and adjacent cells .
4. Limited leakage of cytosolic components = low inflammation.

Question 17

What is blebbing ?

Answer 17

blebbing = A defined feature of apoptosis. Cell cytoskeleton has broken down, causing membrane to bulge/vesicles outwards.

Tubules and vesicles budding of the membrane

Question 18

What are the nuclear changes in apoptosis ?

Answer 18

• Nuclear chromatin condenses on nuclear membrane

- DNA cleavage

Question 19

What are the biochemical changes in apoptosis?

Answer 19

• Expression of charged sugar molecules on outer surface of cell membranes (recognised by macrophages to enhance phagocytosis)
• Protein cleavage by proteases, caspases(family of proteases)

Question 20

What happens if you run a cell treated by apoptosis through an agarose gel ?

Answer 20

Apoptosis causes characteristic laddering of chromosomal DNA

If you run a normal cell you will get one single high molecular weight band

Question 21

What are some specific causes of apoptosis?

Answer 21

1. There is cell death in embryonic hand and this create 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. Death of neutrophils during an acute inflammatory response
5. 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 22

Give some real life examples of apoptosis uses

Answer 22

Metamorphosis -This is the tadpole tail being lost through apoptosis

Interdigital web loss-This is where webbing is lost through apoptosis between the fingers.

Question 23

What are the two types of apoptosis?

Answer 23

Intrinsic and extrinsic

Question 24

What is intrinsic apoptosis?

Answer 24

DNA damage – p53-dependent pathway
Interruption of the cell cycle
Inhibition of protein synthesis
Viral Infection (Once the virus has entered the cell)
Change in redox state

Question 25

What is extrinsic apoptosis?

Answer 25

(Relative to the cell rather than body)
Withdrawal of survival factors e.g. mitogens
Extracellular signals (e.g. TNF)
T cell or NK (Natural Killer) (e.g. Granzyme).

Question 26

What are caspases?

Answer 26

• These are the point of convergence of apoptosis.
• They are cysteine proteases
• They form an activation cascade as one cleaves and activate next

Question 27

Outline Caspase activation

Answer 27

Inactive procaspase Y contains three parts:
N terminus
C terminus

It is cleaved by an upstream caspase in two places which release N terminal pro domain and creates a active caspase Y after rearrangement of small and large subunit.

Question 28

Outline the caspase cascade

Answer 28

There is an imitator caspase (8/9)
These will activate further caspases and then those will activate even more.

Not just caspases , the downstream ones have additional substrates. These are called effector caspases (1,3,6.7)

There is cleavage of cytosolic proteins and cleavage of nuclear proteins (Lamin -Nuclear envelope destruction)

PYRAMID SHAPE _THROUGH SIGNAL AMPLIFICATION

Question 29

What are some substrates for caspases and what do they effect ?

Answer 29

Lamin A/B -Nuclear envelope 
PARP- DNA Repair
DNA-PK-DNA repair
Toposiomerase 2- DNA replication
Raf-1-Signalling
Akt/PKB-Cell survival
STAT1-Signalling
eLF4-Translation

Question 30

What is the effect of caspase activation?

Answer 30

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

Degradation of cytoplasm -Cytoskeleton degradation

Question 31

How do initiator caspases activate themselves?

Answer 31

They will cleave each other when in close proximity

Question 32

Describe the structure of the parts of ligand induced multimerization

Answer 32

It activates extrinsic apoptosis

Process:
1.There is an extracellular ligand which will bind to its receptor via the ligand binding domain on the cell membrane

2.There is a shared domain between the receptor and the death adaptor called the death domain.

3. There is also a shared domain between the death adaptor and procaspase -8 called the death effector domain.
   Finally there is a protease domain

The function of these domains is to allow proteins to bind together to dimerise /multimerise

Question 33

Outline the process of Ligand induced multimerisation using TNF

Answer 33

Tumour Necrosis Factor
1.TNF will bind to the ligand domain on the TNFR and this brings the death domains to close proximity creating environment where dimerisation can take place.

2.There is a death adaptor protein called FADD (FAS-associated protein with Death domain) with high concentration of death effector domains which will recruit Procaspase-8.

This creates a complex called Death-inducing signalling complex (DISC)

We get proteolysis which activates the caspase 8 which can move from membrane to cytoplasm and move into cytoplasm and activate caspase cascade

Question 34

How does intrinsic apoptosis become activated ?

Answer 34

This is through the release of cytochrome c release from the mitochondria

Question 35

What is Cytochrome C ?

Answer 35

It is a mitochondrial matric protein and it becomes released due to oxidative stress which causes it to become more permeable (Permeability transition) .

Question 36

Outline what molecules are involved in Cytochrome C induced apoptosis

Answer 36

There is cytochrome c
There is a death adaptor protein called APAF-1 (Apoptotic Protease Activating Factor )
There is an initiator caspase -Procaspase 9
The caspase recruiter domain (CARD)
Cytochrome c binding site on top of the APAF1

The whole thing is called APOPTOSOME

Question 37

What regulates cytochrome C release from mitochondria ?

Answer 37

Family of protein called BCL-2 which form a pore
Two types:
Anti-apoptotic (bcl-2,bcl-XL) -These repress cytochrome release
Pro-apoptotic(Bax,Bad,Bid)-Facilitate cytochrome c release

Not all membrane proteins
All of them contain a BH3 domain which is used for creating dimers

Question 38

Outline the process of BAX and BCL-2

Answer 38

BAX -Pro apoptotic
Creates a pore which allows cytochrome c to be released

BCL-2 -Anti-apoptotic
It will bind to BAX and cytochrome c will remain inside the mitochondria

BAD - (pro-apoptotic)
BAD will bind to BCL-2 and it will displace it allowing cytochrome c to be released again

Question 39

How does TP53 regulate BCL-2

Answer 39

TP53-
Transcription factor
DNA Damage triggers TP53 expression
Transcription is driven by TP53 driven by BAX(TP53 target protein) and this causes membrane insertion
This will form new pores which can’t be blocked leading to death as more cytochrome c is released

Question 40

How does phosphorylation regulate BCL-2 ?

Answer 40

This is through growth factors
BAD
Survival signals will activate kinase (serine -threonine kinase/Protein kinase B)
This kinase will phosphorylate BAD becomes sequestered in cytoplasm can’t displace BCL-2 meaning cytochrome c can’t be released

Question 41

What happens if survical signals are blocked ?

Answer 41

We do not get activation of PKB meaning we do not get phosphorylation of BAD which can still bind to BCL-2 unblocking the pore and allows cytochrome c to leave and cause cell death .