Unit 4- Control of Cell Cycle & Cell Death

Question 1

How is the cell cycle regulated?

Answer 1

• external signals influence the cell cycle clock
  Anti-Mitogenic signal > enter G0/ quiescent phase
  Pro-Mitogenic signal > enter active cell cycle (G1>S>G2>M)

Question 2

What are the phases of the mammalian cell cycle?

Answer 2

G0- Non-proliferative/ Quiescent phase
G1- Proliferation decision
S- DNA replication
G2- Preparation for mitosis
M- Mitosis

Question 3

What are the cell cycle checkpoints?

Answer 3

Start Checkpoint- is environment favourable > proceed to S phase
G2/M Checkpoint- is all DNA replicated > enter mitosis

Question 4

What does cell cycle progression rely on?

Answer 4

• protein presence
• protein localization (cytoplasm vs nucleus)
• protein activation status (phosphorylated ext)

Question 5

What are cyclins?

Answer 5

• cell cycle proteins that bind to CDKs to activate them/ modulate phosphorylating ability
• undergo cyclic destruction/ synthesis

Question 6

What are the types of cyclins?

Answer 6

G1 Cyclins- bind CDKs at G1/ promote entry into S phase
Mitotic Cyclins- bind CDKs at G2/ promote entry into M

Question 7

What are CDKS?

Answer 7

CDK= Cyclin Dependent Kinase
- phosphorylate substrates on serine/ threonine residues

Question 8

What are CKIs?

Answer 8

CKI = Cyclin-Dependent Kinase Inhibitor
- interfere with Cyclin/CDK complexes (binding/ phosphorylation)

Question 9

What are the 2 families of CKIs?

Answer 9

Cip/Kip- involved in G1 and G1/S control
Ink4- involved in G1/S control

Question 10

How do tumor suppressor/ oncogene mutations cause cancer?

Answer 10

Tumor Suppressor mutation > loss of function (brakes)
Oncogene mutation > gain of function (growth)

Question 11

How is progression through cell cycle checkpoints regulated?

Answer 11

• regulated by CDKs
• depend on regulatory subunit cyclin for activity
• different CDK/cyclin pairs throughout cell cycle > different substrates
  > cyclin levels change rapidly throughout cell cycle (1 direction cycle)

Question 12

What ensures the cell cycle occurs in only 1 direction?

Answer 12

• rapid changes in cyclin levels throughout the cell cycle

Question 13

How are cyclin LEVELS regulated?

Answer 13

• Ubiquitin ligase enzyme promotes ubiquitylation
• adds ubiquitin to phosphorylated cyclins
  > targets cyclin protein for degradation by proteasome
• ensures rapid/ regulated reduction in CDK activity

Question 14

How is cyclin ACTIVITY regulated?

Answer 14

• CDK/cyclin complexes are functionally regulated by CKIs
  INK4- regulate cyclin D complex (G1)
  Cip/Kip- regulate other cyclin complexes

Question 15

How can CKIs regulate growth inhibition/ growth promotion?

Answer 15

• inhibitory factors like TGFβ trigger ↑ expression of CKIs
  > growth inhibition
• mitogens phosphorylate CKIs > phosphorylated CKIs can not translocate to nucleus/ inhibit CDK-cyclin complexes
  > growth promotion

Question 16

What is the period where cells are responsive to mitogenic GFs/ inhibitory TGFβ?

Answer 16

G1 (up until R point)

Question 17

What is the R-point?

Answer 17

Restriction Point- once cell passes, fully commits to cell cycle
- late in G1
- cell commits to a) advance through cell cycle/ b) remain in G1/ c) retreat from active cell cycle into G0
- deregulation of R-point in many cancers

Question 18

What drives cells through the restriction point?

Answer 18

Pocket Proteins = RB Retinoblastoma proteins
- tumor suppressors (lack of functional gene > eye tumors)
- phosphorylation state of pRb influences activity
- hyperphosphorylation of pRB required for transition through R point
- pRb binds to E2F family transcription factors/ inhibit activity

Question 19

How does the phosphorylation status of pRb (pocket protein) influence the cell cycle?

Answer 19

Hyperphosphorylated pRb = inactive/ can not bind and inhibit E2F transcription factors
- cyclin activity leads to ↑ pRB phosphorylation > bypass G1 R-point
- pRb becomes more phosphorylated as cell cycle progresses

Early-mid G1- unphosphorylated > hypophosphorylated
Late GI- hypophosphorylated > hyperphosphorylated

Question 20

How is the R point perturbated in cancer?

Answer 20

• oncogenes promote advancement through restriction point ex) Myc
• tumor suppressors block advancement through restriction point

Question 21

What are the steps in DNA damage repair?

Answer 21

1. DNA lesion detection
2. Recruitment of DNA-repair factors
3. Activation of DNA-repair enzymes
4. Repair
5. Degradation/ inactivation of DNA repair factors

Question 22

What are the potential consequences of DNA damage in cell cycle?

Answer 22

Repair/ Apoptosis/ Senescence/ Mutation

Question 23

What is a drug developed to target the cell cycle?

Answer 23

Palbociclib = selective CD4/6 small molecule inhibitor
- initial attempts to target CDKs failed due to broad actions against all CDKS (poor specificity > high toxicity)
- RB deficient tumors do not respond/ tumors can become resistant

Question 24

What is the difference between Necrosis/ Apoptosis?

Answer 24

Necrosis- pathological/ uncontrolled/ induced by ROS, trauma/ tissue damage/ neighbouring cells affected/ membrane disruption/ cell swells

Apoptosis- controlled cell death/ no inflammation/ no tissue damage/ surrounding cells normal/ induced by specific signaling or cell stress

Question 25

What are 3 apoptosis assays? - caspases are key molecules in apoptosis

Answer 25

Phosphatidylserine/ Annexin V staining - recognition signal for macrophage engulfment TUNEL Assay - addition of labeled nucleotides > nicked DNA - not specific to apoptosis (labels any fragmented DNA) Immunostaining for activated Caspase 3 - requires antibodies that only recognize caspase 3

Question 26

What are the 2 major apoptosis pathways?

Answer 26

Intrinsic (mitochondrial) / Extrinsic (death receptor) - converge at execution phase of apoptosis

Question 27

What are the 2 types of caspases?

Answer 27

Initiator- detect/ transduce signals/ activate effector caspases/ protein-protein interactions Effector = Executioners- cleave downstream targets > cell death

Question 28

What is the caspase paradox?

Answer 28

- caspases cleave substrates at Asp-X-X-Asp - caspase activation requires cleavage at Asp-X-X-Asp

Question 29

What is APAF-1?

Answer 29

- assembles into apoptosome (wheel of death) to activate caspases - several domains including CARD for protein interactions

Question 30

How are apoptosomes formed?

Answer 30

- Cytochrome C interacts with bound ATP, inducing hydrolysis to ADP - results in conformational change in APAF-1 > exposure of CARD domain > interact with each other > complex forms (many APAF-1s come together to form an apoptosome) - ADP/ATP swap allows APAF-1 molecules to assemble > apoptosome by binding ar CARD domains

Question 31

How does caspase activation work?

Answer 31

- procaspase 9 binds via CARD to apoptosome - more CARD bound procaspase 9's recruited > antiparallel orientation - leads to autoactivation of apoptosome-bound caspase 9 > caspase cascade leading to apoptosis

Question 32

How does Cytochrome C induce apoptosis?

Answer 32

- cytochrome c sequestered in intermembrane space of mitochondria - release triggered by pro-apoptotic events > leads to activation of procaspase 9 ext - cytochrome c release also occurs with mitochondrial damage

Question 33

What is the Fas/FasL pathway?

Answer 33

- Fas ligand binds to receptor > assembly of DISC > activation/cleavage of procaspase 8/10 > activation of executioners

Question 34

How is procaspase 8 activated?

Answer 34

- initial ligand-receptor interactions > clustering - recruitment of FADD proteins > large clusters of FAS/FASL > DISC assembly (DISC is like apoptosome) > activate procaspases

Question 35

How is the mitochondrial pathway activated?

Answer 35

- active caspase 8 > cleavage of Bid - truncated Bid > induces cytochrome c release from mitochondria - cytochrome c enhances activation of effector caspases

Question 36

What are the 2 main protein groups of Bcl-2 family?

Answer 36

Pro-Survival > Bcl-2/ Bcl-x - maintain integrity of mitochondrial membrane/ prevent cytochrome c release (protect cells from apoptosis) Pro-Apoptotic> Bax/ Bak/ Bad/ Bim/ Noxa/ Puma - promote cytochrome c release from mitochondria (tumor suppressors)

Question 37

What is the only cytotoxic/pore-forming Bcl-2 protein?

Answer 37

Bax members (Bax/Bak)

Question 38

How do Bcl-2 proteins work/ how do they promote apoptosis?

Answer 38

Bax/Bak = pore-forming (pro-apoptotic) - actively inhibited by Bcl-2/Bcl-x (pro-survival) BH3 Only family (pro-apoptotic Bcl-2's not including Bax/Bak inhibit prosurvival members (Bcl-2/Bcl-x) - inhibition of inhibitors > pro-apoptotic events

Question 39

How are BH3 only proteins regulated?

Answer 39

Bim/ Bid/ Bad > Post-translational mechanisms Puma/ Noxa > De novo expression - gene expression controlled by p53

Question 40

How does p53 contribute to cancer?

Answer 40

- master transcription factor, gene promoters contain p53 binding sites - loss of function of p53 rather than loss of protein (mutations) - pro-apoptotic protein prone to inactivation

Question 41

How does p53 induce apoptosis?

Answer 41

- Fas production > activation of procaspase 8 - Bax production > ↑ release of cytochrome c from mitochondria

Question 42

What is a question about p53?

Answer 42

- is main oncogenic action via interference with p63/p73? (mutant p53 interferes with p63/p73 binding to gene promoters)

Question 43

What is anoikis?

Answer 43

- loss of ECM (cell: matrix) / cell: cell adhesion > metastasis - PKB-AKT signaling has key role in cell death

Question 44

What is the UPR?

Answer 44

- unfolded protein response > to reduce ER stress - if persists can trigger apoptosis/ mutation