Lecture 2

Question 1

What is contact inhibition?

Answer 1

An example of cellular response to anti growth signals released by neighbouring cells

Question 2

What must a cell pass through to enter S Phase?

Answer 2

The Restriction Point

Question 3

What type of complexes are essential for passage through mitosis?

Answer 3

Heterodimeric complexes of a cyclin and a cyclin-dependent kinase

Question 4

What are the the possible cyclin and Ckd complexes? And when in the cell cycle does each act?

Answer 4

CKD1 & Cyclin A: S into G2

CKD1 &Cyclin B: M

CDK4/6 & Cyclin D: Through G1 and passage through restriction point (R)

CDK2 & Cyclin E : R into S

CDK2 & Cyclin A: Early S phase

Question 5

Describe the levels of each type of complex constituent during the cell cycle

Answer 5

CKD levels are fairly constant throughout however, the cyclin level will vary dependent on the stage

Question 6

What are the three CDK regulators?

Answer 6

Cyclins

CDK activating Kinases (CAKs)

CDK Inhibitors(CDKi’s)

Question 7

Which CKDi’s inhibit CDK4? Give an example of a specific inhibitor and how it is normally regulated

Answer 7

The INK4 family

e.g. P15^INK4

Regulated by growth singals: Pro-growth= downregulation and anti-growth= upregulation

Question 8

What other methods can a cell use to control passage through the cell cycle?

Answer 8

Inhibition of complex formation

Proteolysis of complex to stop ammumulation

Question 9

How does TGFß signalling affect the cell cycle?

Answer 9

Inhibits progression out of G1 Phase

TGFß binding to its receptor will activate the intracellular kinase domain and phosphorylate SMAD3. SMAD3 can then complex with SMAD4. This forms a transcriptional complex which activates transcription of a range of CDKi’s and causes cell cycle restraint.

Phosphorylated SMAD3 can also bind E2F; inhibiting its transcriptional activator abilities and respressing Myc expression. With Myc normally causing cell cycle progression.

Two levels of cell cycle restarint.

Question 10

What effect does loss of TGFß signalling have?

Answer 10

Transcription of CDKi’s reduced and expression of Myc increased

Myc will further inhibit CDKi’s

Cell cycle will progress

Question 11

How could cancer cells escape TGFß signalling?

Answer 11

Down-regulation or mutation of the receptor

Loss of SMAD4

Deletion of P15 CDKi

Mutation of CDK4

Overexpression of Myc Oncogene

Question 12

What protein modulates passage through the restriction point? What is this passage called?

Answer 12

Rb Protein

The Rb Switch

Question 13

Where does the name Rb come from?

Answer 13

First seen in childhood Retinoblastoma

Question 14

In what percentage of cancer is the Rb switch disrupted?

Answer 14

Around 90%

Although suspected that is disrupted, in currently unknown ways, in all cancers

Question 15

Why do tumours form in retinoblastoma patients?

Answer 15

Familial Retinoblastoma: Inherited one faulty copy of Rb protein (Germ-line) and loss of second copy will lead to tumour formation (Usually in both eyes)

Sporadic Retinoblastoma: Loss of first copy and then a seperate second mutation causes loss of other copy (Usually only in one eye - becuase less likely)

Question 16

Why is Familial Retinoblastoma seen in the eye?

Answer 16

Although the original mutation is germ-line, and hence in every cell of the body, the exposure to UV light by the retina means it is more likely to develop the second mutation.

These patients do then also have a higher incidence of other cancers in later life due to the germ-line Rb mutation.

Question 17

Describe the phosphorylation state of Rb in different phases of the cell cycle

Answer 17

Unphosphorylated in G0 and early G1

Hypophosphorylated (1 Phosphate group) in Pre-restriction point late G1

Hyperphosphorylated (Severl Phosphate groups) in Post-restriction point late G1

Question 18

Which phosphorylation states indicate active Rb?

Answer 18

Unphosphorylated and hypophosphorylated

Question 19

How does Rb control passage through R?

Answer 19

Regulation of E2F family of transcription factors

Can only bind E2F, and hence influence transcription of genes required for next stages of cell cycle, when not hyperphosphorylated. Unbound E2F then free act without modulation.

Question 20

How does E2F function?

Answer 20

Affects promoter acitivty and influences recruitment of chromatin remodelling factors, e.g. HDACs.

Question 21

What regulates Rb phosphorylation?

Answer 21

Cyclin D-CDK4/6 causes hypophosphorylation

Cyclin E-CDK2 causes hyperphosphorylation

Question 22

Outline the mechanism of Rb phosphorylation by the cyclin-CDK complexes

Answer 22

Hypophosphorylation by cyclin D-CDK4/6 allows some E2F infulenced expression; including expression of cyclin E.

Cyclin E-CDK 2 then causes phosphorylation of more Rb and further activity of E2F, causing more cyclin E production and creating a positive feedback loop; pushing the cell through the restriction point

Question 23

What other effect does Cyclin E-CDK2 have?

Answer 23

Phosphorylates P27^KIP1, an inhibitor of CDK2 and CDK1, marking it for ubiquitination and degradation by the proteosome. Further freeing itself, as well as other cyclin-CDK complexes.

This makes passage through R irreversible; cell cannot go back to state it was in before degradation of the protein and would need to go all the way round the cell cycle again.

Question 24

How can P27^KIP1 be targted by cancer?

Answer 24

In some breast cancer P27 expression is altered or the localisation of the protein can be changed; not sent back to nucleus and instead seen only in cytoplasm.

Question 25

How do DNA tumour viruses target Rb?

Answer 25

Encode proteins, such as E7 in HPV, when inactiavte Rb and flag it for proteolysis. This blocks Rb’s regulation of E2F and leads to unregulated cell proliferation; which is favourable for the virus but causes cancer in the host.

Question 26

What are the genes activated by unbound E2F essential for?

Answer 26

1. Cell cycle progression
2. Replication complex assembly
3. DNA synthesis
4. DNA surveillance
5. DNA Repair
6. Chromatin Structure
7. Mitosis

Question 27

What additional level of control exists beyond the expression of these genes due to E2F?

Answer 27

Many are further controlled by cyclin-CDK complexes.

Question 28

Oultine the roles of cyclin-CDK complexes in the replication complex

Answer 28

Low CDK2 acitivity with cyclin E, in G1, supports replication complex assembly

Rising CDK2 activity with cyclin A, in S, activates the machinery and inactivates assembly proteins

Question 29

Describe the replication complex through G into S phase

Answer 29

In early G1 origin recognition complex (ORC) selects and binds the replication origin. Cdc6, with other proteins, loads helicase (Mcm complex) forming the preRC

In late G1 Cdc6 is inactivated and the preRC is converted to the RC by the addition of polymerases.

In early S phase the RC is activated and the Mcm complex is gradually displaced

Question 30

What are the contributions of the cyclin-CDK complexes?

Answer 30

In early G1 cyclin E-CDK2 supports assembly of Mcm complex.

In late G1 high levels of cyclin A-CDK2 phosphorylates and inactivates Cdc6 and Mcm so they cannot be reused.

In early S phase level of Cyclin A-CDK2 fall and they phosphorylate the DNA polymerases to promote their assembly.

Question 31

What role does Ciz1 play?

Answer 31

Thought to help co-ordinate cyclin E and A function. Swapping them at right time in the cell cycle?