Chapter 7: Apoptosis (Lecture, main)

Question 1

Apoptosis is organized, neat, and tidy, leaving behind little evidence of the preexisting cell. How can apoptosis be so organized, neat, and tidy?

Answer 1

The cell undergoing apoptosis is swept clean during phagocytosis by macrophages.

Question 2

Name physiological processes where apoptosis is important.

Answer 2

• Developmental morphogenesis
• Controlling of cell numbers
• Removal of damaged cells
• Negative and positive selection of lymphocytes
• Cytotoxic effect of radio- and chemotherapy

Question 3

Just read about the differences between apoptosis and necrosis.

Answer 3

Ok

Question 4

So now sum up the six characteristics of apoptosis.

Answer 4

• Cell shrinks
• Membrane blebbing
• Organelles are intact
• Apoptotic bodies
• Chromatin condensation and fragmentation
• No inflammation

Question 5

So now sum up the six characteristics of necrosis.

Answer 5

• Cell swells
• Membranes are leaky
• Organelles are damaged
• Cell lysis
• Chromatin damaged
• Inflammation

Question 6

Depicted in this picture is the morphology of apoptosis in a lymphocyte. Explain the different stages (a-g) seen in this picture (try to describe what you see, furthermore don’t learn this by heart. I think it’s most importantly that you understand the different stages that occur during apoptosis).

Answer 6

(a) the lymphocyte is still a normal cell
(b) the cell has lost volume and its cytoplasmic organelles are now tightly packed and also there’s clumping of chromatin (the black patches you see)
(c) cell blebbing
(d) the chromatin has collapsed down into crescents along the nuclear envelope
(e) the nucleus has collapsed into a black hole (sorry this was the literal description of this figure…)
(f) the collapsed nucleus breaks up into spheres
(g) the cell fragments into apoptotic bodies.

Question 7

Depicted in this picture is a necrotic cell (Fig. a) and an apoptotic cell (Fig b. (cell A)). What are characteristics that you can see in these pictures that are typical for apoptosis or necrosis?

Answer 7

• In Fig. a the cell membrane is damaged and leaky, which is also the case for the organelles.
• In Fig. b (cell A) you can see chromatin rearrangment and condensation and there’s also preservation of membrane and organelles.

Question 8

How can apoptosis be researched in the setting of cancer?

Answer 8

You can stain cancer cells with certain stainings (MGG or Dapi). After this, chemotherapeutic drugs can be applied. A comparison can then be made between cells without these drugs and treated with these drugs, as seen in this picture.

Question 9

What are ways apoptosis can be induced?

Answer 9

• It can be programmed (for example during cell cycle arrest)
• Loss of growth factors or adhesions.
• Activation of death receptors of the TNFR family
• Cytotoxic T lymphocytes (CTLs)
• DNA damage (irradiation, chemotherapy)
• Stress

Question 10

What are four general steps that occur after induction of apoptosis?

Answer 10

(1. Induction of apoptosis)
2. Mitochondrial changes
3. Activation of the caspase family
4. Proteolytic cleavage of structural and functional proteins
5. Induction of apoptosis morphology.

Question 11

Explain the meaning of the words in the sentence that are in bold letters: Caspases are *cysteine-proteases* synthesized as *zymogens*. In order to function they require for an *aspartatic acid at the P1 position*.

Answer 11

• Cysteine-proteases: caspases have specific cysteine protease activity. This means that a cysteine in its active site catalyzes and cleaves a target protein.
• Zymogens: an inactive precursor of an enzyme.
• Aspartatic acid at the P1 position: the cyteine site of the caspase can only cleave another protein after an aspartatic acid residue of the target protein (cleavage then occurs with the cleavage site position P1 of caspase)

Question 12

What caspases are involved in apoptosis?

Answer 12

2, 3, 6, 7, 8, 9 and 10

Question 13

The caspases involved in apoptosis can be divided into initiators, effectors and inflammatory (inflammatory caspases are mostly important in neurodegenerative diseases). Which of the caspases can be divided into these three stages?

Answer 13

• Initiators -> 2, 8, 9 and 10
• Effectors -> 3, 6 and 7
• Inflammatory -> 1, 4 and 5 (so as you can see these inflammatory caspases actually do not play a role in normal apoptosis).

Question 14

In this picture you can see a caspase still in its zymogenic form. In its zymogenic form, four domains are visible. What domains are these? And what is the meaning of QACxG?

Answer 14

• The left (white with gray dots) domain is the pro-domain.
• The dark grey domain is the larger subunit domain.

(- The white domain is a spacer domain)

• The black domain is the smaller subunit domain.
• QACxG is a specific amino acid sequence that is different in all types of caspases. The specific amino acid sequence is important for caspace activation.

Question 15

Caspace activation occurs in two steps. Here it goes from unprocessed caspase to partially processed caspase to fully processed and active caspase. What happens in these two steps?

Answer 15

1. First the smaller subunit domain translocates and binds to the large subunit domain. With this the spacer domain disappears.
2. Second, the pro-domain is cut off. This way, the caspace is activated.

Question 16

What is depicted in this picture?

Answer 16

Caspases cleave at aspartate residues and procaspases are themselves activated by cleavage at aspartate residues. This way, caspases participate in a cascade of activation whereby one caspase can activate another caspase in a chain reaction. This leads to amplification of an apoptotic signal.

Question 17

There are four apoptotic pathways (intrinsic, extrinsic, granzyme B mediated and ER mediated apoptosis pathways). What two are most important?

Answer 17

The intrinsic (stress induced or mitochondrial) and extrensic (death receptor mediated) pathway.

Question 18

For overview purposes:

examine this picture of the intrinsic and extrinsic apoptosis pathway. Please look at the differences between the two.

Answer 18

Ok

Question 19

The extrinsic pathway is regulated by death signals, like Fas and TNF. These ligands bind to their receptor (Fas- and TNF-receptor). What happens after binding of these death signals to their receptors (so in other words; describe the extrinsic pathway)?

Answer 19

Binding causes conformational change and trimerization of the receptors (so three TNF receptors trimerize and three Fas receptors trimerize). This is recognized by adaptor proteins (TRADD and FADD), which leads to aggregation of procaspase-8. Procaspase-8 is activated and turned into caspase-8 (initiator caspase). This initiator caspase initiates a caspase cascade, proteolysis and apoptosis.

Question 20

What are important regulators of the apoptosis pathways (extrinsic and intrinsic)?

Answer 20

Bcl-2 family, C-flip and IAPs (inhibitors of apoptosis proteins)

Question 21

What are inhibitors of the Bcl-2 family? And what are activators of the Bcl-2 family?

Answer 21

• Inhibitors: Bcl-2 and Bcl-XL
• Activators: Bax, Bid

Question 22

How is C-flip able to inhibit caspase-8?

Answer 22

C-flip looks a lot like caspase-8 (it only misses an activation domain). Caspase-8 can only be activated when it forms a homodimer with another caspase-8. But C-flip competes with one of these caspases and when it forms a heterodimer with a caspase-8, there’s inhibition of caspase-8 activation and apoptosis.

Question 23

The intrinsic pathway of apoptosis is triggered by cell stress (e.g. DNA damage). Which protein is the first to act? What happens after (think of what protein interactions are made)?

Answer 23

The BH3-protein Bid is the first to act. Bid binds and activates Bax. Bax undergoes conformational change and oligomerizes on the outer mitochondrial membrane.

Question 24

When one of the BH3-proteins has oligomerized on the outher mitochondrial membrane, pores are formed so that other molecules can leave the mitochondrium. What regulators are released from the intermembrane space?

Answer 24

Cytochrome C and procaspase 9.

Question 25

How is the apoptosome formed during the intrinsic pathway? And what function does it have?

Answer 25

The apoptosome is formed when cytochrome C and procaspase 9 leave the mitochondrial space and form a complex with Apaf-1 (cytochrome C binds with Apaf-1 and recruits procaspase 9 after). The apoptosome that is now formed can activate procaspase-9 which leads to a caspase cascade.

Question 26

Explain the rheostat model of the Bcl-2 family.

Answer 26

This model speaks of the basal state of the Bcl-2 family. Here, there’s a balance between anti-apoptotic and pro-apoptotic Bcl-2 proteins. This basal state can change upon either pro-apoptotic or pro-survival stimulation. The balance then shifts towards a state where either more pro-apoptotic or anti-apoptotic Bcl-2 proteins are present.

Question 27

Explain the anti-apoptotic protein neutralization model.

Answer 27

The BH3-only proteins can be subdivided into sensitizers and activators.

• The sensitizers (Bad, Noxa) are BH3-only proteins that can bind to anti-apoptotic proteins (Bcl-2, Bcl-xL, Bcl-w, Mcl-1).
• The activators (Bim, Bid, Puma) activate pro-apoptotic proteins (Bax and Bak).

To conclude: the anti-apoptotic protein neutralization model states that in order for pro-apoptotic proteins to fulfill their function, sensitizers (Bad, Noxa) need to bind and inhibit anti-apoptotic proteins (like Bcl-2). And also activators (Bim, Bid, Puma) can then bind and activate Bax and Bak.

Question 28

IAPs (Inhibitors of Apoptosis Proteins) act more downstream in the apoptotic pathway and are divided into three classes. What is their main target and through what domain is inhibition of apoptosis achieved?

Answer 28

IAPs mostly inhibit more downstream caspases through their BIR2 or BIR3 domain.

Question 29

How are the extrinsic and intrinsic pathway able to cross-talk/interact with eachother?

Answer 29

Caspase 8 is a typical caspase that is activated during the extrinsic pathway. Active caspase 8 can cleave and activate Bid. Bid can then stimulate the intrinsic pathway of apoptosis by directly activating Bax and Bak, stimulation formation of the apoptosome

Question 30

How will activation of the intrinsic pathway through DNA damage result in cell cycle arrest?

Answer 30

When there’s DNA damage (specifically double strand breaks) the protein ATM (serine/threonine kinase) is activated which will phosphorylate Bid which leads to cell cycle arrest.

Question 31

p53 induces apoptosis in response to DNA damage and cellular stress. Induced apoptosis by p53 can be either transcription-dependent or transcription-independent. Describe the transcription-dependent pathway.

Answer 31

p53 senses DNA damage and induces the expression of genes that code for death receptors and pro-apoptotic members of the Bcl-2 family (this all happens in the nucleus!!). Furthermore it represses the expression of anti-apoptotic factors.

Question 32

By what protein is the transcription-dependent pathway regulated (or: what protein can upregulate p53 in the setting of transcription-dependent apoptosis)?

Answer 32

Puma protein (Bcl-2 family member)

Question 33

p53 induces apoptosis in response to DNA damage and cellular stress. Induced apoptosis by p53 can be either transcription-dependent or transcription-independent. Describe the transcription-independent pathway.

Answer 33

Here, p53 activates Bax in the cytoplasm (!!!) which causes release of cytochrome c and caspase activation.

Question 34

One of the hallmarks of cancer is resisting apoptosis. A normal cell “resists” apoptosis because caspases (for example) are present as zymogen and can’t fulfill their function as zymogens. In many cancers you can see that the immune system is triggered to induce cancer cells for apoptosis by activating caspase-9. But how can these cancer cells evade this apoptotic signal?

Answer 34

In many cancers you can see that caspase-9 is active. Caspase-9 can activate caspase-3 for induction of apoptosis. But in cancer cells XIAP (X-linked Inhibitor of Apoptosis Proteins) is bound to caspase-3, which prevents apoptosis.

Question 35

Name three (general) ways how apoptosis is prevented in cancer cells.

Answer 35

1. Mutations in apoptosis regulating genes
2. Inhibition of pro-apoptotic genes and proteins
3. Upregulation of anti-apoptotic genes and proteins

Question 36

True or false: Most mutations of the apoptotic pathway occur in the extrinsic pathway.

Answer 36

False, most mutations occur in the intrinsic pathway.

Question 37

Match proteins/genes with mutations and with types of cancers (note: all these proteins belong to the intrinsic apoptotic pathway):

• Proteins/genes: p53, Apaf-1, Bcl-2, Bax, XIAP, Survivin
• Mutations: epigenetic inactivation, mutation, translocation/amplification, induction)
• Types of cancers: lung cancer, solid tumors, follicular lymphoma, colon tumor, leukemia, prostate cancer, Head and Neck cancer, diffuse large B-cel lymphoma, lymphoma, metastatic melanoma.

Answer 37

• p53: mutation; solid tumors, leukemia, lymphoma
• Apaf-1: epigenetic inactivation; metastatic melanoma
• Bcl-2: translocation/amplification; follicular lymphoma, diffuse large B-cell lymphoma.
• Bax: mutation; colon tumor
• XIAP: induction; leukemia, lymphoma, lung cancer, prostate cancer
• Survivin: head and neck cancer

Question 38

Mutations can also be found in the extrinsic pathway. Mutations can be located in the Fas receptor or in Caspases 8 and 10. Describe what type of mutations can occur in FasR or in the Caspases and describe what type of cancers results from this.

Answer 38

• Fas receptor: “ordinary” mutation; melanoma and squamous cell carcinoma
• Caspases 8 and 10: epigenetic (loss) or “ordinary” mutations; neuroblastoma and small cell lung cancer.

Question 39

Answer these three questions in short:

1. How do anti-cancer drugs kill tumor cells?
2. Does deregulation of apoptosis in cancer correlate with an aggresive phenotype of cancer?
3. What will happen to cancer cells when the apoptotic pathways are inhibited?

Answer 39

1. By apoptotic activation
2. Yes!
3. They are resistant to anti-cancer treatments.

Question 40

What are three mechanisms that result in chemotherapy-resistance?

Answer 40

1. Mutations in p53
2. Upregulation of anti-apoptotic Bcl-2, IAP family members
3. Downregulation of pro-apoptotic Bcl-2 family members.

Question 41

We can make use of apoptotic proteins and use them as markers for diagnostic and prognostic purposes in tumor biopsies. How can the use of apoptotic proteins as markers help us in rationalized therapies (targeting molecular mechanisms)?

Answer 41

Through the use of apoptotic protein markers we can identify cancer cells that have an apoptotic signal and we can then target these cells. In this way:

• pro-apoptotic factors can be activated.
• anti-apoptotic can be suppressed.

Question 42

One of the mechanisms that induces the activation of the extrinsic apoptotic pathway is through TNF-related apoptosis-inducing ligand (TRAIL). When TRAIL binds to its receptor it recruits Fas-associated death domain (FADD) adaptor protein. FADD has a death effector domain (DED) that recruits a similar DED expressed by procaspase-8 or -10. In cancer, this process is of course turned off. What can be used as treatment for activation of this apoptotic pathway?

Answer 42

Recombinant TRAIL ligand and antibodies against TRAIL receptors (R1 and R2) (this treatment is still in trail (phase 2/3) due to liver toxicity).

Question 43

How can Bcl-2 be inhibited (name 4 ways)?

Answer 43

1. Inhibition of gene transcription
2. Breakdown of mRNA using antisense
3. Inhibition with small-molecule inhibitors
4. Inhibition with endogeneous antagonists.

Question 44

ABT-263 (navitoclax) and ABT-199 (venetoclax) are small molecules that are used for inhibition of Bcl-2. How do these drugs work?

Answer 44

• ABT-263 binds to the same binding site of Bcl-2/Bcl-XL as the pro-apoptotic members of Bad.
• ABT-199 binds to the binding site of Bcl-2 (direct inhibition).

Question 45

Vorinostat (SAHA) is a histone deacetylase (HDAC) inhibitor. What is the function of this drug?

Answer 45

It acts as an endogenous antagonist It inhibits HDAC and acts to induce the expression of epigenetically suppressed genes. In this way it can induce the expression of the pro-apoptotic protein Bid.

Question 46

How can IAPs be inhibited?

Answer 46

1. Breakdown of mRNA using antisense (IAP antisense oligonucleotides)
2. Inhibition with small-molecule inhibitors
3. Inhibition with endogenous antagonists.

Question 47

DNA damage induces apoptosis via:

A. Caspase 9 activation

B. Induction of p53 expression

C. Caspase 3 activation

D. All three of the above mentioned answers are correct

Answer 47

D. All three of the above mentioned answers are correct

Question 48

Briefly explain how triggering of apoptosis in tumor cells can contribute to new therapies against cancer and give an example of a therapy (this was a question in the lecture, so I have no ‘good’ answer)

Answer 48

Tumor cells have developed ways to evade apoptotic signals. If we were to think of a therapy that would trigger apoptosis in tumors (like doing extreme DNA damage, like with chemotherapy) this would help us a lot. An example of a therapy that would trigger apoptosis is using antisense molecules to prevent translation of anti-apoptotic proteins (like Bcl-2).