Foty - apoptosis

Question 1

Syndactly

Answer 1

When apoptosis doesn’t occur in the proper time, your digits are stuck together.

Question 2

Morphological features of apoptosis and ways to detect apoptosis

Answer 2

You can see cell shrinkage, cell blebbing, apoptotic bodies, chromatin condensation.

• You can also see PS flipping, which basically means that usually phosphotidyl serine is found on the inside of the PM. If it is found on the outside then you know there is apoptosis going on. You have to attach a die to the ends of PS lipids though.
• You can also use a technique called TUNEL to look for nicks in the DNA. DNA that is undergoing apoptosis will have nicks in the DNA. You can use a terminal transferase to add a dUTP to the end of 3’ fragments to see the nicks. This has high sensitivity, can be done fast, and can be reproduced fairly easily. But, we don’t know the minimum number of strand breaks, necrotic cells can be false positives, and the detergent that is used makes the cells more fragile.
• You can also look for DNA laddering. Because of the DNA fragments that are made, when ran on a gel, you will see distinct DNA laddering pattern every 180 bp or so. The DNA is cleaved by caspase-activated DNAase.
• Speaking of caspase, you can also look at caspace activation. Inactive caspase is 33 KD, but in order to become active, which happes in cells undergoing apoptosis, the caspase has to be cleaved so you will see 17 KD bands in cells undergoing apoptosis as well as 33 KD bands.

Question 3

Intrinsic and Extrinsic pathways of apoptosis

Answer 3

• Intrinsic is from the mitochondria
• extrinsic is from the death receptors

They converge on the caspases

Question 4

caspases

Answer 4

Caspases are key enzymes that give rise to the morphological changes that occur for apoptosis. They must be cleaved to become active. There are two types of apoptotic caspases:
- initiator caspases - 2,8,9,10
- effector caspases (executioner) - 3,6,7
Caspases are regulated on a post-translational level. They have to have a lot in the cell already so they can act quickly.
- Pro-apoptitic stimulus occurs (Don Corleone) and cleaves the initiator caspase (Clamenza) who then cleaves the effector caspase (Lucabrazzi) who then initiates apoptosis.

Question 5

How does caspase activation result in DNA fragmentnation?

Answer 5

The effector caspases (3 and 7) cleave DFF45 which then cause it to dissociate from DFF40. This activates DFF40 and allows it to oligimerize and activate DNAse. This causes the 180 bp laddering pattern.

Question 6

BCL2 family

Answer 6

regulate the integrity of the mitochondrial membrane. They are pro-apoptotic an anti-apoptotic. There must first be some sort of damage, which then activates p53 and activates the BCL2 proteins. When these BCL2 proteins are activated, the cytochrome c begins to leak out of the mito and caspase 9, which then activates caspase 3,6,7.

Question 7

3 types of cell death

Answer 7

Apoptosis - programmed cell death
Autophagy - self-cannnibalism
necrosis - cell death when we dont want it to happen.

Question 8

Role of mito in intrinsic pathway

Answer 8

mito contain many pro-apoptotic proteins such as cytochrome c, which are released in response to apoptotic signals. Pro-apoptotic molecules normally found in the cytosol translocate to the mitochondrial membrane and stimulate the formation of pores allowing cytochrome c to leak out. Cytochrome c interacts with APAF-1 to form an apoptosome. This activates caspace 9.

Question 9

BH3 proteins involved in apoptosis

Answer 9

The actual names of the proteins aren’t important but know that you have some proteins that are pro-apoptotic and some that are anti-apoptotic and if you have a propensity to express one vs. the other you might have some issues. For your knowledge though (and just in case), Bax and Bak promote pore formation and initiates apoptosis. Bcl-2- binds Bak/Bax and prevents pore formation

Question 10

Bcl-2 and cancer

Answer 10

In follicular lymphoma there is a common translocation that puts the Bcl-2 gene right next to the IgG heavy-chain locus. This causes overexpression of Bcl-2.

Question 11

Summary of intrinsic pathway

Answer 11

• triggerd by p53 in response to DNA or cell damage
• chemo and radiation target p53
• p53 causes transcriptional upregulation of pro-apoptosis members of the Bcl-2 family such as PUMA and BAX
• BAX causes the release of Cytochrome c from the mito, which causes APAF1 to activate caspase 9.
• caspase 9 initiates 3,6,7

Question 12

Extrinsic pathway

Answer 12

DR4 and DR5 are main receptors in this pathway. They are receptive to Apo2L/TRAIL. This activation recruits FADD. This then induces the formation of DISC, which undergoes catalysis. This activates caspase 8 and 10. 8 and 10 then cleave 3,6,7 and leads to apoptosis.

Question 13

Autophagy

Answer 13

The cell cannabilizes itself and recycles the proteins for metabolism. 4 phages - induction, autophagasome formation, autophagasome lysosome fusion, autophagasome breakdown.

• induction - through the mTOR pathway. If a cell is starving there is a signal of autophagy related genes.
• autophagasome formation - the autophagasome will surround whatever parts of the cell need to e chewed up.
• autophagasome-lysosome fusion - fuse the autophagasome with the lysosome

autophagasome breakdown - because of the degradive nature of the lysosome or vacuole, the autophagic body is broken down

Question 14

Differential features of cell death mechanisms

Answer 14

KNOW THE CHART ON THE LAST SLIDE

Question 15

which are pro-apoptotic, antiapoptotic and regulators/sensors

Answer 15

pro - BAX and BAD
anti - BCL2, BCL-XL, MCL1
r/s - BH3 Only proteins - BAD BID PUMA - sense damage and tell others to either undergo apoptosis or not

Question 16

p53

Answer 16

• activate DNA repair proteins
• can cause apoptosis
• arrest scell growth at G1/S check point

It is kept in check by Mdm2. If Mdm2 is released from p53 then it will degrade the cell by apoptosis. Oncogenes also mediate p53 activation, mediated by ARF. p53 is activated by N-terminus phosphorylation. MAPK and checkpoint kinases target transcriptional activation of p53.

when stress occurs, p53 becomes active. It causes increased transcription of p21. This leads to inactive CDK/Cyclin and holds the cell in cell cycle arrest and either repairs DNA or undergoes apoptosis.

Question 17

Convergence of 2 pathways

Answer 17

1) converge at actiation of 3,6,7

2) BID is activated in extrinsic and it will tie up anti-apoptotic in intrinsic thus helping the process along

Question 18

When cell damage occurs, what happens that causes the cell to be degraded by apoptosis? Intrinsic pathway

Answer 18

One of the BH3 proteins, BAD BID or PUMA, will bind to BL-2 anti-apoptotic proteins (BCL-2, BCL-XL, MCL1), which then allow BAX and BID to be active. When this happens, Cytochrome c will leak out of the membrane.