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Cancer > Apoptosis > Flashcards

Apoptosis Flashcards

1
Q

Define Necrosis.

A

Unregulated cell death associated with trauma, cellular disruption and an inflammatory response

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2
Q

Define Apoptosis.

A

Regulated cell death; controlled disassembly of cellular contents without disruption – no inflammatory response

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3
Q

Describe the process of necrosis.

A
  1. plasma membrane becomes more permeable
  2. cell swells and the membrane ruptures
  3. release of proteases leading to dissolution and auto-digestion of the cell
  4. localised inflammation
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4
Q

What are the two phases of apoptosis? Describe them.

A

Latent phase
- Death pathways are activated, but cells appear morphologically the same

Execution phase:

  1. Loss of microvilli and intercellular junctions
  2. Cell shrinkage
  3. Loss of plasma membrane asymmetry
    - (phosphatidylserine lipid appears in outer leaflet)
  4. Chromatin and nuclear condensation
  5. DNA fragmentation
  6. Formation of membrane blebs`(bulge/protrusion)
  7. Fragmentation into membrane enclose apoptotic bodies (these are then taken up by macrophages)
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5
Q

What is an important feature of apoptosis that distinguishes itfrom necrosis?

A

Plasma membrane remains intact – no inflammation

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6
Q

What DNA modification is seen during apoptosis?

A
  1. Fragmentation of DNA ladders (seen in agarose gel)

2. Formation of more ends, which are labelled by adding an extra fluorescently-labelled tag in a TUNEL assay

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7
Q

What other types of cell death are there other than necrosis and apoptosis?

A
  1. Apoptosis-like PCD - some, but not all, features of apoptosis
  2. Necrosis-like PCD - “Aborted apoptosis”
    NOTE: cell death is GRADED
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8
Q

What are caspases?

A

Cysteine-dependent aspartate-directed proteases
They are the executioners of apoptosis
They are activated by cleavage

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9
Q

Which caspases are effector caspases?

A

3, 6 and 7

-carry out apoptosis

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10
Q

Which caspases are initiator caspases?

A

2, 8, 9 and 10

- trigger apoptosis

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11
Q

Describe the structure of effector caspases.

A

They are single chain polypeptides consisting of a small and large subunit
The subunits are released by proteolytic cleavage

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12
Q

Describe the structure of initiator caspases.

A

They have the same two subunits (p20 and p10) found in effector caspases but they also have a targeting subunit (protein-protein interacting domain)

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13
Q

What are the two types of targeting subunit that initiator caspases can have?

A

CARD – caspase recruitment domain

DED – death effector domain

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14
Q

How are active caspases formed?

A

Cleavage of inactive procaspases is followed by the folding of 2 large and 2 small chains to form an active L2S2 heterotetramer

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15
Q

What are the two mechanisms of caspase activation?

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

2. Death by default (mitochondrial (intrinsic) death pathway)

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16
Q

Describe the structure of death receptors.

A

consist of:

  1. Cysteine-rich extracellular domain
  2. Transmembrane domain
  3. Intracellular tail with a death domain (DD)
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17
Q

What are the two important adaptor proteins in the death by design pathway (extrinsic) and how are they different?

A

1.FADD – positive regulator that promotes cell death
– FADD= DED (death effector domain) + DD (death domain)
2. FLIP – negative regulator that inhibits the death pathway and allows regulation
- FLIP = DED + DED

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18
Q

Describe signalling of apoptosis through Fas.

A
  1. Fas ligand binds to Fas receptor (on CTLs) and the Fas receptors undergo trimerisation, which brings the three DDs together
  2. The trimerised DDs recruit FADD, which binds via its own DD
  3. FADD then recruits and oligomerises procaspase 8 through the DED of procaspase 8
  4. Binding of procaspase 8 to FADD forms DISC (death-induced signalling complex)
  5. DISC formation results in cross-activation of procaspase 8
  6. Active caspase 8 is released, which then activates effector caspases
19
Q

Describe the important of oligomerisation in this pathway.

A
  1. Some initiator caspases have intrinsic low catalytic activity
    - oligomerisation brings them close enough together to allow transcleavage
  2. others are activated by conformational change on oligomerisation
  3. at least 2 procaspases are required to form an active caspase
20
Q

Describe how FLIP acts as an inhibitor of apoptosis

A

Death receptor activation of procaspase 8 is inhibited by FLIP (negative regulator)

  • It has two DED domains and can compete with procaspase 8 to bind to the DED domains of FADD
  • FLIP incorporates into the trimer but it has NO proteolytic activity so cannot cleave the other procaspases
21
Q

As an overview, describe death by default (mitochondrial, intrinsic pathway).

A
  1. Cellular stresses e.g lack of growth factor
  2. Loss of mitochondrial
    membrane potential (ΔΨ)
  3. Release of cytochrome C
  4. Formation of the
    apoptosome complex
22
Q

What does the apoptosome consist of?

A

(wheel of death)

  1. APAF-1 (apoptotic activating factor 1)
  2. Cytochrome C
  3. ATP
  4. Procaspase 9
23
Q

Describe the domains found within APAF-1.

A
  1. CARD domain
  2. ATPase domain
  3. WD-40 repeats (protein-protein interactions)
24
Q

Explain fully, how death by default leads to caspase activation.

A
  1. Cytochrome C released from the mitochondria bind to the WD-40 repeats of APAF-1, form a heptamer (apoptosome) which requires ATP
  2. CARD domain binds to CARD on procaspase 9
    (so seven procaspases can bind to one apoptosome)
  3. Oligomerisation brings multiple procaspase 9s close together allowing them to cross-cleave
  4. Resulting in cleavage, activation and release as active caspase 9 tetramer, which initiates a caspase cascade leading to apoptosis
25
Q

What pro-apoptotic protein links the death by default and death by design pathways? Explain how it works.

A 
Bid  
Caspase 8 (generated by the death by design pathway) cleaves Bid, which travels to the mitochondrion and promotes the release of cytochrome C – thus triggering the mitochondrial death pathway
26
Q

How can energy levels of a cell show whether a cell is going through apoptosis or necrosis?

A

Apoptosis requires energy whereas necrosis does not
- intrinsic mitochondrial pathway requires energy (ATP), and apoptosis will always use this pathway to some extent so will always use ATP

27
Q

What is an important family of proteins that act as intrinsic modulators of apoptosis?

A

Bcl-2 family

28
Q

There are three main groups of Bcl-2 proteins. What is common to all three groups?

A

BH3 domain – this is a dimerisation motif, which allows members of the family to form dimers with each other

29
Q

What are the anti-apoptotic Bcl-2 proteins and where are they found?

A

Bcl-2
Bcl-xL
They are found localised on the mitochondrial membrane

30
Q

What are the pro-apoptotic Bcl-2 proteins and where are they found?

A 
Bid 
Bad 
Bax 
Bak 
These are found in the cytoplasm and in the mitochondrial membrane
31
Q

Other than Ras signalling, what other pathway does growth factor binding to growth factor receptors activate?

A

PI3-kinase

32
Q

What type of molecule is PI3-K?

A

Lipid kinase

33
Q

What are the main subunits of PI3-K?

A

Adaptor subunit
Targeting subunit
Catalytic subunit

34
Q

What is the main action of PI3-K?

A

PI3-K converts PIP2 to PIP3

35
Q

What effect does this action of PI3-K have that leads to inhibition of apoptosis?

A
  1. PIP3 is recognised by the adaptor subunit of Protein Kinase B (PKB/Akt)
  2. This allows PKB to move to the cell membrane where it becomes activated
  3. PKB phosphorylates and inactivates Bad ( a pro-apoptotic protein)
36
Q

Describe the arrangement of the anti-apoptotic and pro-apoptotic proteins when growth factor signalling and the PI3-K pathway is active.

A

This means PI3-K can produce PIP3 – so PKB/Akt is activated meaning that Bad is phosphorylated and inactivated
Bad is held in an inactive heterodimer with 14-3-3
On the mitochondrial membrane, Bak and Bax are held in inactive heterodimers with Bcl-2 and Bcl-xL

37
Q

Describe how loss of growth factor signalling can lead to apoptosis.

A
  1. This means loss of activation of the PI3K pathway (as GF absent) so less PIP3 produced so less activation of PKB/Akt
  2. Bad gets dephosphorylated and dissociated from its inactive heterodimer with 14-3-3
  3. Bad then moves to the mitochondrial membrane and binds to the anti-apoptotic proteins (Bcl-2 and Bcl-xL) via its BH3 domain
  4. This displaces the pro-apoptotic Bcl-2 family proteins ( Bax and Bak) from their inactive heterodimers
  5. So Bax and Bak then form a pore in the mitochondrial membrane allowing the release of cytochrome C from the mitochondrion – this leads to apoptosis
38
Q

Summarise the effects of PKB/Akt in promoting cell survival.

A
  1. Phosphorylates and inactivated Bad ( a Bcl-2)
  2. Phosphorylates and inactivates caspase 9
  3. Inactivates FOXO transcription factors (FOXOs promote the expression of apoptosis-promoting genes)
  4. Stimulates ribosome production
39
Q

Name two extrinsic regulators of apoptosis and describe their actions.

A

PTEN

  • Lipid phosphatase
  • Counteracts the activation of PKB
  • Reduces cell survival and promotes apoptosis

IAPs (Inhibitor of Apoptosis proteins)

  • Binds to procaspases and prevents their activation
  • Can bind to activate caspases and inhibit their activity
40
Q

Are the following tumour suppressor genes or oncogenes?

a. Bcl-2
b. PTEN
c. PKB/Akt

A

a. Bcl-2
Oncogene –over expression of Bcl-2 results in cancer (cancers are anti-apoptotic)

b. PTEN
Tumour suppressor gene –as inactivation raises the PI3-K pathway and thus allows increased survival of cancer

c. PKB/Akt
Oncogene- over expression of PKB leads to increased cell survival thus cancer

41
Q

What does apoptosis deal with?

A
  1. Harmful cells egg DNA damaged
  2. Developmentally defective cells egg self antigen B cells
  3. Excess cells e.g get rid of webbing during embryonic development of hands
  4. Obsolete cells e.g mammary epithelium at end of lactation
  5. Exploitation e.g chemotherapeutic killing of cells
42
Q

What are the mechanisms of apoptotic cell death?

A
  1. The executioners – Caspases
  2. Initiating the death programme
    - Death receptors
    - Mitochondria
  3. The Bcl-2 family
  4. Stopping the death programme
43
Q

What are the three main possible cytoprotective/anti-apoptotic pathways?

A
  1. Bcl-2, Bcl-xL: intrinsic pathway
  2. FLIP, IAPs (inhibitor of apoptosis proteins): extrinsic pathway
  3. growth factor pathways via PI3’-K and PKB/Akt

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