1st February

Question 1

Within the cell cycle, in which phase is the restriction point located?

Answer 1

G1

Question 2

During which phase is the cell responsive to extracellular growth factors?

Answer 2

G1

Question 3

Which Cdk-cyclin complexes control passage through the restriction point?

Answer 3

Cdk4/6 - Cyclin D

Question 4

Why are the growth phases of the cell cycle important in adult cells?

Answer 4

Give the cell time to respond to both internal and external factors.
Prevents the cell going into mitosis with DNA damage
Ensures the cell has enough mass to create 2 new cells

Question 5

Outline the process of pre-replicative complex formation

Answer 5

1. During early G1 2 large helicases bind to the origin of replication in their inactive form by Cdc6, Cdt and ORC
2. In S-phase the Cdk2 activates the pre-RC by phosphorylating specific initiator proteins. DDK also phosphorylates specific helicase subunits.
3. The helicases unwinde the DNA, replication starts and the pre-RC leaves the DNA
4. Mitosis occurs
5. Repeat

Question 6

When is ORC bound to the origin of replication?

Answer 6

Throughout the cell cycle

Question 7

What prevents the pre-RC reassembling on the DNA after replication?

Answer 7

APC/C is inactivated in late G1 helping prevent further pre-RC assembly later in the cycle
The 1st stage of pre-RC assembly can only occur without Cdks present

Question 8

What prevents the pre-RC ‘firing’ before S phase?

Answer 8

It requires Cdk2 activity therefore the presence of cyclin E is required which only occurs during S phase

Question 9

How does Cdk2 prevent the assembly of the pre-RC?

Answer 9

It phosphorylates Cdc6 triggering it for destruction

Question 10

What is the function of Rb?

Answer 10

It suppresses cyclin E function through the recruitment of HDAC

Question 11

Outline the mechanism of activating cyclin E expression

Answer 11

OFF - E2F1 is bound to the promoter, to which Rb binds. This recruits a HDAC which deacetylates the promoter –> heterochromatin meaning that the gene can not be transcribed

ON - Cdk4/6 phosphorylates Rb meaning that HDAC can no longer bind. This means HAT can come and acetylate the DNA –> expression of cyclin E. Once cyclin E has been produced it creates a positive feedback loop as the Cdk2-cyclin E complex further phosphorylates Rb

Question 12

How is cyclin E expression under the control of GFs?

Answer 12

Cyclin D is required for activation of Cdk4/6 which is required for the phosphorylation of Rb to relieve the inhibition on the cyclin E gene.

Cyclin D is under the direct control of GFs

Question 13

Name 3 examples of proteins in the Rb family

Answer 13

Rb
P107
P130

Question 14

Name 2 CKIs acting on Cdk2-cyclin E

Answer 14

p27

p21

Question 15

Is p21 subject to internal or external cues?

Answer 15

Internal cues such as cell stress and DNA damage

Question 16

Is p27 subject to internal or external cues?

Answer 16

External cues such as growth signals and anti-mitogenic factors

Question 17

Can p21 and p27 bind to CDK4/6 - Cyclin D complexes?

Answer 17

Yes however they can not inhibit it, causing CDK4/6-Cyclin D to sequester the CKIs

Question 18

Outline the mechanism behind regulation of p27 stability

Answer 18

p27 is phosphorylated by p27
Phosphorylated p27 is ubiquitinated by SCF E3 Ub ligase
It is the polyubiquitinated and sent to the 26s proteasome for degradation

Question 19

How is p27 stability increased during G1?

Answer 19

During G1 APC/C degrades SCF preventing p27 degradation which prevents the premature activation of Cdk2

Question 20

Which cdk does the INK4 family inhibit?

Answer 20

Cdk4

Question 21

List the members of the INK4 family

Answer 21

p14, p16, p18 and p19

Question 22

How do INK4 family proteins inhibit CDK4?

Answer 22

They bind to the CDK preventing cyclin binding

Question 23

How are p15 levels affected by TGF-beta signalling?

Answer 23

They are increased

Question 24

Give examples of some of the ways in which G1/S control is lost in cancer

Answer 24

Inherited Rb mutations
Inherited p16 mutations
Acquired Rb/p16 mutations 
Epigenetic silencing of p16 through promoter methylation
Reduced expression of p27 
Cyclin D OE

Question 25

What form of cancer do inherited Rb mutations lead to?

Answer 25

Retinoblastoma

Question 26

What form of cancer do inherited p16 mutations lead to?

Answer 26

Familial melanomas

Question 27

List some examples of targeted approaches of treatment aimed at the restriction point

Answer 27

anti-HER2 receptor MoAbs
anti-HER1, HER2, HER4 Tyrosine kinase inhibitors
RAS farnesyltransferase inhibitors
RAF inhibitors
MEK inhibitors
mTOR inhibitors

Question 28

Are mice lacking Cdk2 viable?

Answer 28

Yes

Question 29

What is the only essential substrate of Cdk4/6?

Answer 29

Rb

Question 30

What is observed when mice have all 3 cyclin D genes KO?

Answer 30

Die at mid/late stage gestation due to severe anaemia indicating that hematopoeitic cells are cyclin D dependent
Organs develop normally indicating that other cells are not cyclin D dependent

Question 31

How does Akt aid CDK activation

Answer 31

It relieves 2 constraints:

Akt –I GSK3beta preventing it from phosphorylating and destabilising cyclin D

Akt –I FOXO TFs preventing them from producing p27Kip1 and P21Cip1