What is contact inhibition?
An example of cellular response to anti growth signals released by neighbouring cells
What must a cell pass through to enter S Phase?
The Restriction Point
What type of complexes are essential for passage through mitosis?
Heterodimeric complexes of a cyclin and a cyclin-dependent kinase
What are the the possible cyclin and Ckd complexes? And when in the cell cycle does each act?
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
Describe the levels of each type of complex constituent during the cell cycle
CKD levels are fairly constant throughout however, the cyclin level will vary dependent on the stage
What are the three CDK regulators?
CDK activating Kinases (CAKs)
Which CKDi's inhibit CDK4? Give an example of a specific inhibitor and how it is normally regulated
The INK4 family
Regulated by growth singals: Pro-growth= downregulation and anti-growth= upregulation
What other methods can a cell use to control passage through the cell cycle?
Inhibition of complex formation
Proteolysis of complex to stop ammumulation
How does TGFß signalling affect the cell cycle?
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.
What effect does loss of TGFß signalling have?
Transcription of CDKi's reduced and expression of Myc increased
Myc will further inhibit CDKi's
Cell cycle will progress
How could cancer cells escape TGFß signalling?
Down-regulation or mutation of the receptor
Loss of SMAD4
Deletion of P15 CDKi
Mutation of CDK4
Overexpression of Myc Oncogene
What protein modulates passage through the restriction point? What is this passage called?
The Rb Switch
Where does the name Rb come from?
First seen in childhood Retinoblastoma
In what percentage of cancer is the Rb switch disrupted?
Although suspected that is disrupted, in currently unknown ways, in all cancers
Why do tumours form in retinoblastoma patients?
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)
Why is Familial Retinoblastoma seen in the eye?
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.
Describe the phosphorylation state of Rb in different phases of the cell cycle
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
Which phosphorylation states indicate active Rb?
Unphosphorylated and hypophosphorylated
How does Rb control passage through R?
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.
How does E2F function?
Affects promoter acitivty and influences recruitment of chromatin remodelling factors, e.g. HDACs.
What regulates Rb phosphorylation?
Cyclin D-CDK4/6 causes hypophosphorylation
Cyclin E-CDK2 causes hyperphosphorylation
Outline the mechanism of Rb phosphorylation by the cyclin-CDK complexes
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
What other effect does Cyclin E-CDK2 have?
Phosphorylates P27KIP1, 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.
How can P27KIP1 be targted by cancer?
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.
How do DNA tumour viruses target Rb?
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.
What are the genes activated by unbound E2F essential for?
- Cell cycle progression
- Replication complex assembly
- DNA synthesis
- DNA surveillance
- DNA Repair
- Chromatin Structure
What additional level of control exists beyond the expression of these genes due to E2F?
Many are further controlled by cyclin-CDK complexes.
Oultine the roles of cyclin-CDK complexes in the replication complex
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
Describe the replication complex through G into S phase
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
What are the contributions of the cyclin-CDK complexes?
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.
What role does Ciz1 play?
Thought to help co-ordinate cyclin E and A function. Swapping them at right time in the cell cycle?