13.3 Apoptosis I

Question 1

What is apoptosis?

Answer 1

apoptosis is programmed cell death.

Question 2

Why is normal cell death essential?

Answer 2

b/c it plays a role in balancing the growth of new cells and it also plays important roles in some stages of life in embryogenesis.

Question 3

What is the faith of cells with irreparable DNA damage?

Answer 3

normally these cells undergo apoptosis to avoid becoming cancerous.

Question 4

Reduced apoptosis is thought to be important for?

Answer 4

for development of tumors and resistance to chemotherapy.

Many cancer therapies interfere w/DNA replication, which leads to activation of apoptotic signaling pathways, but if these pathways become dysfunctional through mutations, cancer cells develop resistance to anti-cancer drugs as a result.

Question 5

Defects in apoptosis are linked to?

Answer 5

autoimmune diseases

cells of the immune system are screened for cells that might produce antibodies against targets in our own bodies and if detected, cells are instructed to kill themselves. If this process is disrupted cells that recognize targets in our own bodies can escape cell death and this leads to autoimmune disorders

Question 6

Excessive apoptosis is involved in?

Answer 6

neurodegenerative diseases and in part of the tissue destruction that occurs after vascular occlusions of the heart and brain (heart attack, stroke)

Question 7

What is necrotic cell death?

Answer 7

cell death that does not involve active participation of that cell. Necrotic cell death is uncontrolled and the cell doesn’t participate in its own death, and just ends up dying for some reason.

Question 8

What is the difference between necrotic and apoptotic cell death?

Answer 8

NECROTIC: uncontrolled
APOPTOTIC: controlled and highly conserved series of internal signaling events and morphogenetic steps

NECROTIC: cell does not participate in its own death, cell just dies for some reason
APOPTOTIC: cell is actively involved in the death process

Question 9

Apart from uncontrolled and cell participation, what other differences exist between apoptosis and necrosis?

Answer 9

APOPTOSIS: Cell shrinkage
NECROSIS: cell swelling

APOPTOSIS: membrane integrity maintained
NECROSIS: membrane integrity lost

APOPTOSIS: role of mitochondria and cytochrome c
NECROSIS: no role of mitochondria

APOPTOSIS: no leak of lysosomal enzymes
NECROSIS: leak of lysosomal enzymes

APOPTOSIS: characteristic nuclear changes
NECROSIS: nuclei lost

APOPTOSIS: apoptotic bodies form
NECROSIS: do not form

APOPTOSIS: DNA cleavage
NECROSIS: no DNA cleavage

APOPTOSIS: Activation of specific proteases
NECROSIS: no activation

APOPTOSIS: regulated process
NECROSIS: no regulated

APOPTOSIS: evolutionarily conserved
NECROSIS: not conserved

APOPTOSIS: do not induce inflammation responses
NECROSIS: induces inflammation responses

Question 10

What are the different experimental ways to detect apoptosis?

Answer 10

1) staining cells with antibodies that detect activated elements of apoptotic signaling pathway
2) Run DNA isolated from cells out on a gel
3) TUNEL Staining

Question 11

Describe how running DNA isolated from cells out on a gel helps detect apoptosis

Answer 11

DNA is isolated from cells and placed on a gel. The random digestion of DNA linkages between nucleosomes leaves the DNA wrapped around an individual nucleosome intact. The result are pieces of DNA that have a specifically defined length. It’s long enough to wrap around one nucleosome, or two, if for some reason the linkage between two nucleosomes was not cut, or three if two links escape cutting and so on, and you get a laddering effect where you see DNA at varying predictable sizes on the gel

Question 12

What is TUNEL staining

Answer 12

This method can be used to detect apoptotic cells in tissue or in situ, without having to extract the DNA and running it on a gel. and allows one to see the distribution of apoptotic cells.

The method labels the end of DNA molecules w/a marker which can be recognized by an antibody. Cells that have intact DNA have only a few DNA ends, essentially it has two ends for each chromosome (we have 46 chromosomes so really only 90 ends in an individual cell).

Cells undergoing apoptosis have millions of DNA ends as a result of the nuclease activity that chops up the DNA and this results in very BRIGHT signals from those cells.

Question 13

What is one of the major hallmarks of apoptotic cell deat?

Answer 13

early destruction of a cell’s DNA. This occurs through the activation of nucleases which degrade the links of DNA between nucleosomes, which are the parts of DNA wrapped around histones.

Question 14

What is an advantage of using TUNEL staining over DNA extraction for apoptosis detection?

Answer 14

In TUNEL you do not have to extract DNA and you can look at tissues to see distribution of apoptotic cells.

Question 15

What does Caspase stand for and what are their roles?

Answer 15

Caspase stands for “Cysteine Aspartate Specific Proteases” and they mediate apoptosis by cleaving target proteins at specific aspartic acid residues and forming a cascade of activity to amplify the effects of activating the apoptotic pathway.

Question 16

How are caspases activated?

Answer 16

activated by proteases which cut them at defined placed.

They’re produced in cells in an inactive form, which is called procaspase. Cutting off the pro domain and cutting the resulting peptide at one internal site, often by another caspase leads to the formation of a dimer and then an active tetramer.

Question 17

The caspase cascade consist of caspases that are activated early and some that actually cut up pieces of the cell. What are these known as?

Answer 17

1) Initiator caspases - activated early in the process

2) Executioner caspases - cut up the pieces of the cell

Question 18

Which caspases are initiator caspases ?

Answer 18

Caspases 2, 8, 9 and 10

Question 19

Which caspases are executioner caspases?

Answer 19

Caspases 3, 6, and 7

Question 20

What happens once the caspase cascade has been initiated?

Answer 20

this process is tightly regulated and proceeds in a series of steps. once started, the process of apoptosis is irreversible and so much of the control involved in preventing apoptotic cell death is going to be at the level of prevent the activation of the initiator caspases

Question 21

What tow pathways can bring about cell death through apoptosis?

Answer 21

1) Extrinsic (receptor mediated) pathway

2) Intrinsic (mitochondrial) pathway

Question 22

Cells that signal from from the outside for apoptosis use what kind of receptors?

Answer 22

Cell surface receptors known as death receptors.

Question 23

What is a type of death receptor?

Answer 23

the Fas system. Where the Fas ligand binds to the death receptor.

Question 24

What happens when binding occurs between Fas ligand to the death receptor?

Answer 24

it leads to clustering and auto-activation of the initiator procaspase-8, which leads to the initiation of the downstream caspase cascade.

Question 25

What kind of signaling cascade does the intrinsic apopototic pathway use?

Answer 25

it involves a signaling cascade using mitochondria

Question 26

What role does mitochondria play in the apoptotic intrinsic pathway?

Answer 26

mitochondria releases cytochrome C into the cytoplasm, which serves as a signal to initiate apoptosis and this process is regulated by members of a family of proteins called Bcl1-family

Question 27

What regulates mitochondria in the intrinsic pathway?

Answer 27

proteins called BcI2-family

Question 28

In extrinsic pathway, what happens when Fas lingand binds to FasR and activates FasR?

Answer 28

Binding leads to clustering of the Fas receptors. The activation of FasR leads to recruitment of an adaptor protein called FADD.

Question 29

What does FADD stand for?

Answer 29

Fas-associated death domain

Question 30

What is the role of FADD, which pathway does it play a role?

Answer 30

in the extrinsic pathway.

FADD has a death domain that binds to the Fas receptor and an effector domain that recruits procaspase-8 or sometimes 10 to the receptor complex. This results in the formation of a Death Inducing Signaling Complex (DISC)

Question 31

What happens when initiator caspases are brought close together in the extrinsic apoptotic pathway? What does it do to the DISC?

Answer 31

the initiator caspases slowly cut target sequences on each other, even though they aren’t active, which then leads to activation of the other protein and even if only a small number of the procaspases that are brought to a DISC are activated, the activated caspases then go about activating all the other procaspases in the DISC very efficiently and rapidly, and so it’s easy for this whole cascade system to start up once it’s formed.

Question 32

How does is the intrinsic pathway initiated?

Answer 32

it forms a cluster of initiator procaspases and then the initiator procaspases activate each other to start the cascade.

Question 33

What differs between the intrinsic and extrinsic pathway?

Answer 33

the intrinsic pathway has no ligand binding or receptor clustering. Only procaspases cluster.

Question 34

What occurs after initiator procaspases activate each other in the intrinsic pathway and start the cascade for apoptosis?

Answer 34

The mitochondria releases a small inter-membrane protein called cytochrome C into the cytoplasm and there it activates a protein called Apaf1

Question 35

What does Apaf1 stand for? what activates this protein?

Answer 35

Apaf1 stands for apoptotic protease activating factor 1. and it is activated by cytochorme C, which is released by the mitochondria

Question 36

What is the role of Apaf1 and what pathway uses this protein?

Answer 36

Intrinsic pathway uses Apaf1.

Apaf1 is able to form oligomers through interaction w/other Apaf 1 proteins using what’s called a caspase recruiting domain or CARD.

Question 37

What happens with CARD under normal conditions?

Answer 37

under normal conditions, the CARD domain is held in the cytochrome C binding region of Apaf, however, binding of cytochrome c to Apaf releases the CARD, which then allows Apaf to form clusters.

Question 38

What releases CARD from Apaf1?

Answer 38

binding of cytochrome C to Apaf

Question 39

Apaf’s that form clusters are called?

Answer 39

Heptamer, since the cluster has seven members.

The functional name is apoptosome

Question 40

What does the apoptosome do?

Answer 40

it recruits initiator procaspase-9 and then procaspase-9 will cluster and activate each other and initiate the caspase cascade

Question 41

What steps are involved in the intrinsic pathway of apoptosis?

Answer 41

1) initiator procaspases form a cluster and activate each other
2) mitochondria then releases cytochrome C into the cytoplasm
3) Cytochrome C activates Apaf1
4) Binding of Apaf with cytochrome C releases CARD (caspase recruiting domain)
5) Apaf1 can form oligomers with interaction w/other Apaf proteins using CARD. SO once CARD is released, Apaf forms clusters.
6) The cluster becomes a functional apoptosome
7) Apoptosome recruits initiator procaspase-9 and this procaspase 9 will cluster and activate each other and initiate the caspase cascade

Question 42

What steps are involved in the extrinsic pathway of apoptosis?

Answer 42

1) Fas ligand binds to FasR and leads to clustering of the Fas Receptor
2) Activation of FasR leads to recruitment of adaptor protein FADD
3) FADD has a death domain that binds to the Fas receptor and an effector domain that recruits procaspase-8 or 10 to the receptor complex.
4) This results in formation of a death inducing signal complex (DISC)
5) Small number of procaspases that are brought to a DISC are activated , the activated caspases then go about activating all the other procaspases in DISC very rapidly
6) this activates caspases such as procaspase-3 to induce apoptosis

Question 43

Which pathway is receptor mediated?

Answer 43

the extrinsic pathway

“death receptor”

Question 44

What type of stimuli can activate the intrinsic mitochondrial pathway?

Answer 44

DNA damage, treatment of cells w/chemotherapeutic agents, growth factor withdrawal, and reactive oxygen species