Cancer 9 - Apoptosis

Question 1

Why do cells undergo apoptosis (programmed cell death - PCD)

Answer 1

1. Harmful cells
2. Developmentally defective cells
3. Excess/unnecessary cells
4. Obsolete cells
5. Exploitation

Question 2

What are the differences between necrosis and apoptosis

Answer 2

Necrosis = unregulated cell death, associated with trauma, cellular disruption and INFLAMMATORY RESPONSE

Apoptosis = regulated cell death, controlled disassembly of cellular contents without disruption, NO INFLAMMATORY RESPONSE

Question 3

Explain how necrosis happens (localised inflammation)

Answer 3

1. Plasma membrane becomes permeable (e.g. trauma)
2. Chromatin condenses, fluid rushes in –> cell swells and cellular membrane ruptures
3. Release of proteases –> autodigestion and dissolution of cell
4. Cell lysis, invasion of phagocytic cells, inflammation

Question 4

What are the 2 phases of apoptosis and what happens in each phase

Answer 4

1. Latent phase - death pathways activated but cell remains morphologically the same
2. Execution phase - Loss of microvilli and intercellular junctions
   - cell shrinkage
   - loss of plasma membrane asymmetry (phosphatydylserine appears in outer leaflet/membrane)
   - Chromatin and nuclear condensation
   - DNA fragmentation
   - Formation of membrane blebs
   - fragments enclosed into membrane enclosed apoptotic bodies

Question 5

Apoptotic bodies are phagocytosed by….?

Answer 5

Macrophages

Question 6

Why is there no inflammation in apoptosis?

Answer 6

Because the cellular contents is not released so no mediators are attracted

Question 7

As the cell shrinks in apoptosis, what closes around it? Why

Answer 7

Epithelium closes around it to prevent entry of things such as pathogens via the lumen

Question 8

How can apoptotic nuclei be detected

Answer 8

1. DNA ladders - show fragmentation as DNA is being broken down
2. Adding fluorescent tagged base - DNA fragmentation = more ends = brighter nuclei show more fragmentation

Question 9

Aside from necrosis and apoptosis, what are the other forms of cell death

This is a graded response

Answer 9

1. Apoptosis-like PCD = has some but not all features of apoptosis - phagocytic recognition molecules displayed before plasma lysis
2. Necrosis-like PCD - variable features of apoptosis before cell lysis - “aborted apoptosis”

Question 10

What do caspases (Cysteine-dependent Aspartate-directed Proteases) do?

Answer 10

They degrade everything inside the cell - “the executioner”

They are activated by proteolysis - trigger cascade of activation

Question 11

What are the 2 types of caspases

Answer 11

1. Initiator caspases - these are triggered first.
   2 domains:

CARD (Caspase Recruitment Domain) (cascades 2, 9)

DED (Death Effector Domain) (cascades 10, 8) - these have homotypic protein-protein interactions, provide scaffolding

2. Effector caspases - these do the chopping up (caspases 3,6,7)

Question 12

How does caspase maturation happen

Answer 12

Zymogens (procaspases) undergo proteolytic cleavage –> removes the pro-domains and it separates to form 2 distinct long (L) and short (S) subunits –> which then forms an active L2S2 hetero-tetramer

Question 13

Which 3 things do caspase cascades involve

Answer 13

1. Amplification
2. Divergent responses
3. Regulation

Question 14

How do initiator cascades trigger apoptosis?

Answer 14

By cleaving and activating effector caspases (which carry out the apoptotic programme)

Question 15

What 4 actions can effector caspases have

Answer 15

(Monomeric substrates)

1. Inactivation
2. Activation

(Multiprotein complexes)

1. Disassembly
2. Release

Question 16

Give an example of a nuclease that is activated by effector caspases

Answer 16

Caspase-Activated-DNase (CAD)

Question 17

Give an example of a protein/complex being cleaved and inactivated by effector caspases

Answer 17

Nuclear lamins cleaved - leading to nuclear breakdown

Question 18

What are the 2 mechanisms of caspase activation

Answer 18

1. Death by design - receptor mediated (extrinsic) pathways

2. Death by default - mitochondrial (intrinsic) pathway

Question 19

Describe death receptors

Answer 19

1. They are transmembrane, cysteine-rich extracellular domains that are trimerised

Question 20

What do intracellular death domains attract

Answer 20

Adapter proteins

Question 21

Name and describe the roles of 2 adapter proteins in receptor-mediated apoptosis

Answer 21

1. FADD - has a DED and DD domain - responsible for activating apoptotic path
2. FLIP - 2x DED domain - inhibits apoptotic path

DD and DED domains bind to similar domains on other proteins

Question 22

Explain how signalling via death receptors occurs in the Fas/L

Answer 22

1. Fas receptor trimerised - due to FasL on lymphocyte
2. FADD (adapter protein) recruited via its DD to the DD of Fas
3. Recruitment and oligomerisation of procaspase 8 (via its DED to FADD DED) —> triggers DISC (Death-Inducing Signalling Complex)

Question 23

How many procaspases are needed to form an active tetramer

Answer 23

2

Question 24

Which 2 ways can activate procaspase 8

Answer 24

1. Transcleavage
2. Conformational change upon oligomerisation

(active procaspase is released when procaspase 8 interacts with DED which causes cleavage)

Question 25

What are the two types of FLIP?

Answer 25

Small and long FLIP - they have the same homology in the DED domain as the procaspases but they have no proteolytic activity - so the FLIPs must compete with the procaspase to inhibit activation of caspase 8 Essentially FLIP inhibits procaspase 8 activity by binding onto the DED domain of the Fas/FADD receptor tail - no proteolytic compartment so no transcleavage

Question 26

Describe the intrinsic pathway of apoptosis (mitochondrial regulation)

Answer 26

1. Loss of mitochondrial membrane potential due to cellular stress 2. Release of Cytochrome C and other apoptosis inducing factors into cytoplasm 3. Formation of the apoptosome complex

Question 27

What are the constituents of the apoptosome complex

Answer 27

Apoptotic activating factor-1 (APAF-1), procaspase 9, ATP, Cytochrome C

Question 28

Describe the role(s) of Apaf-1 in the apoptosome | Apaf-1 is a scaffolding protein to organise the apoptosome

Answer 28

1. Has CARD domains at the centre of the wheel to recruit caspases 2. Has WD-40 repeats at C terminal - which binds to cytochrome c

Question 29

When stimulated, the apoptosome is a ........meric structure

Answer 29

Heptameric

Question 30

How are the caspases involved in the apoptosome

Answer 30

Procaspase 9 binds (docks) onto the CARD, and forms a dimer with caspase 9 in the middle of the wheel - potentially 7 procaspase 9s can bind as well (heptameric) Oligomerization brings many procaspase 9s together - which results in cleavage and activation --> release of active caspase 9 tetramer --> initiating the caspase cascade ---> which triggers apoptosis

Question 31

What is an essential requirement for the apoptosome

Answer 31

ATP

Question 32

What may determine whether cell death is by necrosis or apoptosis

Answer 32

Energy levels in the cell

Question 33

How may the extrinsic and intrinsic apoptotic pathway be linked

Answer 33

The extrinsic pathway, upon activation of caspase 8, activates Bid proteins which stimulates the intrinsic (mitochondrial) pathway by stimulating release of mitochondrial proteins into cytoplasm. Caspase 8 and apoptosome then go onto activate caspase 3

Question 34

Which family of proteins modulates apoptosis?

Answer 34

Bcl-2 | some have Transmembrane domains

Question 35

How many groups of Bcl-2 proteins are there?

Answer 35

3 Group 1 Group 2 Group 3 - contains BH3 only (BH3 = dimerisation motif)

Question 36

Which are the anti-apoptotic proteins

Answer 36

Bcl-2, Bcl-xL (hooked on mitochondria)

Question 37

What are the pro-apoptotic proteins

Answer 37

Bid, Bad, Bax, Bak (move between cytosol and mitochondria) Group 2 and 3

Question 38

Which 2 pathways does activation of the PI3 receptor (Growth factor receptor) cause PI3 kinase stimulated by growth factors (e.g. EGF, insulin)

Answer 38

1. ERK pathway (growth) | 2. PI3 kinase pathway (survival, proliferation)

Question 39

What is PI3 kinase and what is it involved in

Answer 39

It is a lipid kinase (2 subunits - P110 and P85) It is involved in growth control and cell survival

Question 40

Which anti-apoptotic protein kinase does PI3 kinase activate

Answer 40

PKB/Akt (via PIP3) PI3 kinase converts PIP2 --> PIP3

Question 41

What is P13 kinase blocked by? What effect does this have

Answer 41

Blocked by PTEN Reduces cell survival

Question 42

4 ways in which PKB/Akt induces cell survival by blocking apoptosis (intrinsic pathway)

Answer 42

1. Phosphorylates and inactivates Bad 2. Phosphorylates and inactivates caspase 9 3. Inactivates FOXO Tfs (FOXOs promote expression of pro-apoptotic genes) 4. Other, e.g. stimulates ribosome production and protein synthesis

Question 43

Explain how apoptosis is regulated by BH3 dimerisation

Answer 43

1. Usually (GF present), Bad is inactive and phosphorylated in cytosol 2. Bcl-xL/2 on mitochondria and form inactive dimers via BH3 GFs absent 3. Bad dephosphorylated and released 4. Displaces Bcl-2xL dimer 5. Bax/Bak form a pore in the mitochondrial membrane, out of which cytochrome c leaks out 6. Apoptosis triggered

Question 44

What is PTEN?

Answer 44

Lipid phosphatase Means that we can alter lipid levels in the cell to control apoptosis/survival

Question 45

Which proteins can regulate extrinsic apoptosis

Answer 45

Inhibitor of Apoptosis Proteins (IAPs) - They bind to procaspases and prevent activation - bind to active caspases and inhibit their activity

Question 46

Name anti-apoptotic pathways for intrinsic pathway

Answer 46

Bcl-2, Bcl-xL

Question 47

Name anti-apoptotic pathways for the extrinsic pathway

Answer 47

FLIP, IAPS

Question 48

Name 2 therapeutic uses of apoptosis

Answer 48

1. Kill harmful (oncogenic) cells - e.g. virally infected cells, cells w DNA damage 2. Chemotherapeutic killing of tumour cells (e.g. dexamethasone stimulates DNA cleavage)