Fotey - Cell Death

Question 1

Autophagy

Answer 1

Catabolic repurposing of cell materials - SELF CANNIBALISM

Question 2

Necrosis

Answer 2

Premature death by external factors - ACCIDENTAL DEATH

Question 3

Morphological Markers of Apoptosis

Answer 3

• electron-dense nucleus
• nuclear fragmentation
• intact cell membranes
• disorganized cytoplasmic organelles
• large clear vacuoles
• surface “blebs” (trix), cell shrinkage
• loss of cell-cell adhesion
• apoptotic bodies

Question 4

Biochemical markers of apoptosis (3)

Answer 4

• PS Flipping: Phosphatidyl serine flips to exterior membrane, can use immunocytochemistry and flow cytometry to identify
• TUNEL: identifies nicks in DNA present during apoptosis - faster, higher sensitivity (but doesnt distinguish necrotic cells)
• DNA Laddering: consistent DNA fragment sizes based on caspace-activated DNAase cleaving (CAD)

Question 5

Caspace Activation

Answer 5

cleaves from 33kda to 17 when activated, so on western blot, can identify the apoptotic cell by product at 17

Question 6

Caspaces

Answer 6

“Hitmen”, cleaved to become active.
2 types: initiators (2,8,9,10) and effectors (3,6,7).
pathway: initiator activator–>initiator–>effector–>apoptosis

Question 7

How does caspace activation cause DNA fragmentation?

Answer 7

effector caspaces cleave DFF40/45 dimer (DFF = DNA Fragmentation Factor), they separate, DFF40 dimerizes with itself and becomes DNAase, cleaves DNA, causes apoptosis

Question 8

Intrinsic Apoptosis Pathway

Answer 8

DNA damage sensed by p53.
-BCL2 family has pro and anti-apoptosis members:
pro: BAX + BAK
anti: BCL2, BCL-XL, MCL1
balance between them determines if apoptosis proceeds
-BH3 proteins (Bad/Bid/Puma) are sensors,damage activates them to activate pro-apoptotic factors by inhibiting BCL2
-BAX/BAK activation causes cytochrome c leakage from mitochondria, activating APAF1 forming apoptosome, activating caspaces (starting with 9)…–>apoptosis

Question 9

p53

Answer 9

Activate DNA repair in response to damage

• arrests growth at G1/S point
• initiates apoptosis
• Cellular p53 levels normally low via 1) Mdm2 ubiquitination and degradation and 2) Mdm2 transport of p53 out of nucleus (preventing transcription)

Question 10

P53 activation

Answer 10

N-terminal phosphorylation by MAPK (JNK1-3, ERK1-2, p38 MAPK) or Checkpoint Kinases (ATR, ATM, CHK-1,2, CAK) or oncogenes (ARF).
-inactivates Mdm2 association, so free p53 causes increased p21, inactive Cdk/cyclin, holding cell cycle for DNA repair or causing apoptosis

Question 11

BCL2 in cancer

Answer 11

Follicular Lymphoma: BCL2 overexpression can prevent Bid/Bad/Puma from inhibiting BCL2, preventing apoptosis when necessary, causing cancer

Question 12

Extrinsic Apoptosis Pathway

Answer 12

INDEPENDENT OF P53
Pro apoptotic Ligand (Apo2L or TRAIL) binds Death Receptor 4/5 –> recruits FADD–>form DISC complex, recruits Procaspaces 8,10–>cleaved to Caspaces 8,10, –> activates caspaces 3,6,7–>apoptosis

Question 13

Convergence of Extrinsic/Intrinsic Pathways

Answer 13

1) Convergence at initiator caspaces

2) Procaspaces 8/10 of extrinsic pathway activate Bid–>inhibit BCL2–>activate BAX/BAK–>cyt c. release…

Question 14

Autophagy Mechanism

Answer 14

-used as housekeeping recycling, cannibalism in starvation
Mechanism:
1) Induction: Autophagy is inhibited by mTor, but mTor inactivated by starvation, causing autophagy gene transcription
2) Autophagosome Formation: form membrane around targeted portion of cell
3) Autophagosome-lysosome fusion
4) Autophagosome breakdown:

Question 15

Necrosis Mechanism

Answer 15

irreversible, caused by external factors

Characteristics: exploded plasma membrane (unlike apoptosis),

Question 16

Differences between apoptosis, necrosis, autophagy:

Answer 16

see slide 32 of Foty - Cell Death lecture

Question 17

Apoptosis

Answer 17

Programmed cell death - SUICIDE

-critical for preventing syndactyl by promoting cell death between digits (failure causes webbing)