Session 7 - The real cell cycle

Question 1

Outline the stages of cell cycle

Answer 1

G0 – Senescence
G1 – Growth of cell
S phase – DNA increases from 2n to 4n. Generates non-diploid number of chromosomses
G2 – Reorganised interphase cromatin into chromosomes.
M – Mitosis occurs, with multiple phases within (pro met anna on the telephone). DNA transitions from 4n to 2n.

Question 2

What are the three main checkpoints of the cell cycle?

Answer 2

G1 checkpoint - pRb
G2 checkpoint - p53
M checkpoint

Question 3

How can we detect the phases of the cell cycle?

Answer 3

Flow cytology allows us to detect the phases of the cell cycle by measuring amount of DNA in cell at any one time

Question 4

What is the main protein involved at the G1 to S phase checkpoint

Answer 4

pRb

Question 5

What stimulates cell cycle through pRb checkpoint

Answer 5

Hormones, growth factors and cell contact

Question 6

What are the three main proteins involved in regulation of pRb?

Answer 6

Cyclins
Cyclin dependent serine/threonine kinase
Cyclin dependent kinase inhibitors

Question 7

What does phosphorylation of pRb do?

Answer 7

Inhibits it

Question 8

What does hypophosyphorylation of pRb do?

Answer 8

Activates it

Question 9

What are four mechanisms that regulate cyclin activity?

Answer 9

• Association with cyclins
• Association with CDK inhibitors
• Inhibitory phosphorylation (P21)
• Activating phosphorylation

Question 10

What cyclins act at what points?

Answer 10

• G1 – Cyclin D
• G1/S – Cyclin E
• S – Cyclin A
• M – Cyclin B

Question 11

What are the two mechanisms of action of pRb?

Answer 11

Prevents cell cycle progression via production of HDAC1 which prevents DNA transcription by causing chromatin condensation, and by not releasin transcription factor E2F

Question 12

What do CDKs do to pRb?

Answer 12

If CDKs are active, they will phosphorylate pRb (the master brake), preventing it from inhibiting the release of the transcription factor E2F. When E2F is released it stimulates transcription which allows the cell cycle to progress (by stimulating production of Cyclin E and Cyclin D, as well as DNA polymerase)

Question 13

What does HDAC1 do?

Answer 13

HDAC1 protein activated by pRb causes deacetylation and condensation of chromatin, decreasing transcription. This occurs when pRb is not phosphorylated.

Question 14

What does active pRb do?

Answer 14

Active pRb – E2F production inhibited, HDAC1 activated -> Cell cycle halted

Question 15

What does inhibited pRb do?

Answer 15

Inhibited pRb – E2F activated, HDAC1 inhibited -> Cell cycle stimulated

Question 16

How does p53 moderate cell cycle (with regards to checkpoint 1)

Answer 16

P53 also moderates cell cycle at G1, by blocking cycle progression until DNA repair is completed. It does this by triggering hypophosphorylation of pRb which prevents release of E2F and thus prevents progression of cell cycle.

Question 17

What is the exact mechanism of pRb inhibition?

Answer 17

Requires phosphorylation via CDK4 (cyclin D attached) followed by further phosphorylation by CDK2 (cyclin E attached).

Question 18

What is the exaction mechanism of CDK inhibition?

Answer 18

P16(INK4A) is responsible for the binding to CDKs and preventing their interaction with pRb. This effect is mimicked by p21, p15.

Question 19

What is the wnt signalling path?

Answer 19

Growth stimulating pathway

Question 20

What is the cell surface receptor for WNT?

Answer 20

Frizzled receptor

Question 21

What is activated when WNT ligand binds with frizzled?

Answer 21

Phosphoprotein dishevelled activatied

Question 22

What are the two pathways of WNT?

Answer 22

Canonical and non-canonical

Question 23

What does WNT path do?

Answer 23

Causes accumulation of B-catenin in cell cytoplasm via inhibition of destruction pathway. B-catenin translocates to nucleus, activating cyclin D1 and D2 transcription, and represses p16. This promotes entry into S phase.

Question 24

How is WNT path upregualted?

Answer 24

Increased expression of Wnt ligand proteins or B-catenin causes activation of canonical path and thus activation of cell cycle.

Question 25

What is the purpose of the G2/M checkpoint?

Answer 25

DNA replication check point, found after DNA synthesis

Question 26

What is G2/M regulated by?

Answer 26

Mediated by MPF (maturatin promoting factor). This is a protein composed of Cyclin B and CDK1.
This protein phosphorylates multiple substrates to trigger entry into mitosis.

Question 27

What can MPF be inhibited by?

Answer 27

This can be inhibited by double strand breaks (ATM produced which activates WEE1 to phosphorylate MPF and inhibit it) or DNA damage (ATR -> WEE1 -> MPF phosphorylation).

ATM and ATR also activate P53, which activates CKI P21 and further inhibits cell cycle.

Question 28

What occurs in ataxia telangictasia?

Answer 28

ATM and ATR can be mutated which means this checkpoint does not work, leading to replication of cells with DNA damage.

Question 29

What does CDK2NA do?

Answer 29

Activates pRb AND p53.

Question 30

What are two apoptotic pathways?

Answer 30

Intrinsic and extrinsic pathway

Question 31

What do intrinsic and extrinsic path converge on?

Answer 31

Capse 3

Question 32

Outline the extrinsic path of apoptosis

Answer 32

TNF-a, TRAIL bind to membrane (trail-R) and cause capsase activation and cell apoptosis. This is regulated by p53, which moderates productions of death receptors and capsases.

Question 33

Outline the intrinsic path of apoptosis

Answer 33

Apoptosome is formed by cytochrome C and APAF-1 -> This can only form as a result of increased permeability of mitochondria, and a result of Bcl-2, Bak and Bax (all triggered by P53!).

Question 34

What is required for the intrinsic path to proceed?

Answer 34

For intrinsic pathway to proceed via >mitochondrial permeability, pro-survival BCL-2 family must inhibited and BAX or BAK must be activated (both of these things done by P53!)