Cell Death

Question 1

What is the function of cytochrome P450 ?

Answer 1

• Many compounds not excretable, need to be conjugated into something excretable
• Oxidising them with P450 then conjugating them with something else to make them more soluble and hence excretable

Question 2

Distinguish apoptosis from necrosis.

Answer 2

APOPTOSIS

• May be pathological or physiological
• Single cells
• Biochem events: Energy-dependant fragmentation of DNA
• Cell membrane integrity maintained
• Morphology: cell shrinkage and fragmentation
• No inflammatory response
• Dead cells phagocytosed by neighbouring cells

NECROSIS

• Always pathological
• Groups of cells
• Biochem events: Abnormal ion homeostasis
• Cell membrane integrity lost
• Morphology: Cell swelling and lysis
• Inflammatory response present
• Dead cells phagocytosed by inflammatory cells
• Causes include ischaemia, trauma, metabolic

Question 3

Identify the main kinds of necrosis, where each occurs, and the appearance of each.

Answer 3

Coagulative necrosis: In most tissues. Firm pale area with ghost outlines

Colliquative necrosis: In brain. Dead area liquefied.

Caseous necrosis: In tuberculosis. Pale yellow semi-solid material.

Gangrenous necrosis: necrosis with putrefaction (following vascular occlusion or certain infections). Black

Fibrinoid necrosis: microscopic feature in arterioles in malignant hypertension

Fat necrosis: following trauma (mass) or pancreatitis (multiples white spots)

Question 4

Is programmed cell death the same as apoptosis?

Answer 4

No. Apoptosis is morphological whilst PCD is about intent.

Question 5

Give examples of PCD.

Answer 5

• Embryology – lumen of tubes
• Response to growth signals – menstrual cycle
• Inflammation – resolution, death of neutrophils
• Immune defence – T and Natural Killer cell responses
• Tumour prevention – prevent mutation
• Autoimmune disease – self destruct
• HIV AIDS – HIV and activated T cell death

Question 6

Describe the main molecules involved in DNA fragmentation in apoptosis.

Answer 6

Caspases fragment DNA
Caspases fragment Poly (ADP ribose) polymerase (PARP) which is a repair enzyme
Caspases also fragment Inhibitor of Caspase activated DNAase=ICAD (results in the activation of CAD which “breaks up DNA during apoptosis”)

Question 7

How does PARP relate to cancer treatment ?

Answer 7

In cancer, anticancer drugs/radiation try to damage DNA. If damage DNA, can chose to die by apoptosis but doesn’t die often because of repair enzymes

PARP inhibitors ineffective on their own but given with agents to damage DNA, show real potential, prevent repair (replicate what the cell is doing when it decides to do apoptosis)

Question 8

How does recognition of cells undergoing apoptosis by macrophages and non-professional phagocytes take place ?

Answer 8

After fragmentation, membrane flips inside which means on the outside (outer leaflet), expose Phosphatidylserine which can be recognised by macrophages (recognise membrane bound fragments with abnormalities)

Question 9

What are the two pathways to apoptosis ? Describe the main features of each.

Answer 9

EXTRINSIC

• Trigger comes from outside. Typically through “death” receptors, which transduce messages inside cell
• T cells may trigger this

INTRINSIC
-Oxidative stress (may be metabolic, poisoning, energy failure, DNA damage and p53)

Question 10

What are possible triggers of the extrinsic pathway of apoptosis ?

Answer 10

Cytokines or ligand binding (eg ligand on cytotoxic T cell)

Question 11

Explain the role of cytokines in triggering the extrinsic pathway of apoptosis.

Answer 11

TNF family (produced by macrophages in tumours) is ligand found on cytotoxic T cells.
Binds to CDC95 (=Fas) receptor
Receptor then trimerises
Death domain gets exposed which results in cascade of procaspases which amplify one another (activated by fragmentation), eventually leading to caspapases (which then cleave nuclear proteins, cytoplasm etc.)

Question 12

Explain the role of T cells in triggering the extrinsic pathway of apoptosis.

Answer 12

Cytotoxic T cell contain a couple of things:

1. Granzyme (kind of like a caspase), which activates caspase. Directly introduce granzyme into target cell, it activates caspases. Fragments the cells.
2. Perforin, which perforates cell membrane

Combo of those two results in fast track to destruction through cytoplasmic activation

Question 13

Identify examples of pathological mechanisms in which T cell mediated extrinsic apoptosis is involved.

Answer 13

Viral infection

Transplantation rejection

Question 14

Describe the main features of the intrinsic pathway of apoptosis.

Answer 14

• Triggered by cellular stress, specifically mitochondrial stress caused by factors such as DNA damage and heat shock.
• Upon receiving the stress signal, the proapoptotic proteins in the cytoplasm, BAX and BID, bind to the outer membrane of the mitochondria to signal the release of the internal content. BAK, another proapoptotic protein that resides within the mitochondria, is also needed to fully promote the release of cytochrome c and the intramembrane content from the mitochondria
• Following the release, cytochrome c forms a complex in the cytoplasm with ATP and Apaf-1, an enzyme.
• Following its formation, the complex will activate caspase-9, an initiator protein. In return, the activated caspase-9 works together with the complex of cytochrome c, ATP and Apaf-1 to form an apoptosome, which in turn activates caspase-3, the effector protein that initiates degradation.
• Besides the release of cytochrome c from the intramembrane space, the intramembrane content released also contains apoptosis inducing factor (AIF) to facilitate DNA fragmentation

Question 15

What are the components of an apoptosome ?

Answer 15

A heptameric apoptotic protease activating factor 1 (Apaf-1)-cytochrome-c complex.

Question 16

Identify the function of the apoptosome.

Answer 16

The apoptosome recruits and activates caspases that cleave intracellular substrates and ultimately lead to cell death by apoptosis.

Question 17

What is the physiological response to radiation damage to DNA (possibly due to oxygen free radicals) ? Which part of this response may be affected in cancer ?

Answer 17

p53 activated (sensory of damage in DNA) and forms tetromer.
Makes 3 classes of genes: 
1. stop cell cycle, 2. increase expression and function of DNA repair enzymes and 3. increase expression of genes leading to apoptosis

P53 is affected in cancer

Question 18

Identify examples of pro-apoptotic factors and anti-apoptotic factors.

Answer 18

Pro-apoptotic factors: BAX

Anti-apoptotic factors. Bcl2

Question 19

Explain importance of balance between pro an anti apoptotic factors in influencing apoptosis.

Answer 19

Members of the Bcl2 family dimerise. These dimers allow for control of sensitivity of cells to death (e.g. DNA damage to B cell will often lead to aptopotsis whereas in neurons will not usaully lead to apotpsosis).

BCL2 dimers: stops cytochrome C leaking out of mitochondria (prevents apoptosis)

BAX dimers: strong death signal (lets cytochrome C leak out, hence forming apoptosome and resulting in apoptosis)
E.g. some genes activated by p53 will be pro-death (through extra activation of Bax)

Question 20

What is the normal function of Bcl2 ? Hence, what is a potential effect of abnormal Bcl2 expression ?

Answer 20

Bcl2 keeps cell alive so if overexpressed, a cell that should die does not die and becomes cancerous

Question 21

How is abnormal expression of Bcl2 generally due to ?

Answer 21

This may occur in translocation of promoter of one gene on coding bit of bcl2, resulting in overexpression of bl2, and leading to dimerisation.

Question 22

Does Bcl 2 result in increased proliferation ?

Answer 22

No, just stops cells dying

Question 23

Which pathologies may abnormal Bcl2 expression be found in ? Is there any treatment available for this ?

Answer 23

Typically in follicular lyphoma, B cell malignancy

Treatment: Anti BCL2 therapy (binds to Bcl2 molecules)

Question 24

What are the different ways in which cell death is regulated ?

Answer 24

• Caspases
• Bcl2 and Bax expression
• p53
• Inhibitor of apoptosis=IAP (mop up caspases): need to be able to bring caspases in apoptosome to generate high enough concentration of caspases to overcome inhibitors
• Anti-IAPs: block IAPs

Question 25

Where did the name caspase come from ?

Answer 25

C because cysteine in active site asp because cleaves after aspartate ase because it is a protease

Question 26

Identify some of the molecules cleaved by caspases, and the result of each cleavage.

Answer 26

Cleave - ICAD: destroy genetic information - PARP: prevents DNA repair - lamin: breaks down nuclear architecture - keratin: breaks down cytoplasmic architecture

Question 27

Describe the process of activation of caspases.

Answer 27

"Dimerization and often oligomerisation of pro-caspases, followed by cleavage into a small subunit and large subunit. The large and small subunit associate with each other to form an active heterodimer caspase"

Question 28

What are cancers due to, with regards to intrinsic and extrinsic pathways of apoptosis ?

Answer 28

Cancers due to failure or activation of both intrinsic and extrinsic pathways, because intrinsic and extrinsic pathways communicate (If start chain of events from outside, inside will also contribute).

Question 29

Explain the significance of survival factors in apoptosis, providing an example.

Answer 29

- Survival factors (e.g. growth factors) are extrinsic - If gets signals from inside telling it to die (eg BCL2) but also survival factor from outside telling it to grow (growth factor), will decide not to die - E.g. Hepatitis B/C first cause of liver cancer in the world: kills liver cells but also leads to chronic inflammation which brings in growth factors which, compete against factors requesting for apoptosis to happen, and thereby lead to proliferation (resulting in cancer)

Question 30

How does nutrition relate to apoptosis ?

Answer 30

With higher caloric intake, increased number of growth factors (so less likely to get rid of cells when you need to)

Question 31

Give examples of apoptosis gone wrong.

Answer 31

* Autoimmune disease (apoptosis when do not require it) * Cancer (no apoptosis when require it) * Neurodegeneration (apoptosis when do not require it e.g. dementia)

Question 32

What components of the apoptosis pathway may be targeted in the following pathologies ? - Lymphoma - Cancer - Alzheimer's

Answer 32

Lymphoma: Bcl2 (decrease it) Cancer: IAP (decrease it) Alzheimer's: Caspase 3 (decrease it)

Question 33

What is Pyroptosis ?

Answer 33

A modification of necrosis and apotposis (features of both), exploited by one particular organism through innate (non-specific) receptors.

Question 34

How is pyroptosis triggered ?

Answer 34

Microbial trigger eg Salmonella

Question 35

What are examples of features of apoptosis and necrosis in pyroptosis ?

Answer 35

– Caspase 1 activation, not caspase 3 – Nuclear fragmentation but not cytoplasmic blebbing – Pro-inflammatory

Question 36

What types of receptors are involved in pyroptosis ?

Answer 36

Innate (non-specific) Pattern recognition receptors | – NOD like and Toll like receptors

Question 37

What is Anoikis ? Is it most similar to necrosis or apoptosis ?

Answer 37

Death after losing contact with basement membrane/extra cellular matrix. It has apoptosis morphology.

Question 38

What occurs when anoikis fails ?

Answer 38

Metastasis