L14/15 - Check Point Controls Flashcards
Getting genetic material from one cell to the daughters depends on
Faithful replication of a cells geneome
Proper allocation of the cells DNA to the daughter cells
Cells are only allowed to proceed with the cell cycle if …
If the pre-requisites have been completed sucessfully
What happens if the pre-requistes have not been met
Halt to further advance until the problems have been adressed
The monitoring of cells are known as
Check point controls
What is the result of a cells genome being in need of repair
The cell will not be able to enter S phase
What hapeens if DNA is damaged
Replication will be paused
What happens if the G2/M checkpoint if there is DNA damage
Entry into M phased is blocked
When is a cell allowed to enter anaphase
When all of its chromosomes are properly alligned on the mitotic spindle during metaphase
What monitors each step of the cell cycle
Surveillance mechanisms
What happens if the pre-requiste steps havent been met
Halt to further advance until problems have been adressed
What must be the state of the genome for the cell to be able to proceed into S phase
Genome is not in any need of repair
When may DNA replication be paused
If there is damage to the DNA
If cell needs to pause replication of the DNA what is the effect of this on the duration
Can double the time taken for the genome to be replicated
What must be completed for a cell to proceed through G2 and M phase
Replication of the DNA
M phase entry will be blocked if
DNA damaged
A cell is not permitted to enter anaphase until ….
All of its chromosomes are properly assembled on the spindle during metaphase
How does the increased mutability of the genome provide cancer cells with a prolfierative advantage
Incompatible with normal cell cycle progression
Checkpoint controls block advance through the cell cycle if DNA has been damaged
In addition to activating oncogenes and inactivating tumour supressor genes what else may be inactivated
One or more check point controls
pRB ….
Blocks advance through the cell cycle
What is the molecular goveronor of the R point
RB (retinoblastoma)
Cells are only able to go through the R point only if …
pRb becomes hyperphosphorylated
pRb phosphorylation is governed by
Components of the cell cycle clock
pRb becomes phosphorylated in what phase …
What complex mediates this
D-CDK4/6
Early and mid-G1
Levels of what cyclin increase at the R point
E
What complex mediates the hyperphosphorylation of pRB
E-CDK2
Unphosphorylated/hypophosphorylated pRB is able to bind to
E2F
When hyperphosphorylated what happens to Rb association with E2F
Dissociates
Describe the effect of pRb binding to E2F, and when does this occur
In early and mid G1 - pRb binds to E2F preventing the trasncription of E2F dep genes
pRb becomes hyperphosporylated at the R point - effect of this
Release of E2F
Transcription of genes which mediate the transition between G1 ans S phase
Why is the activity of E2F shortlived
Cyclin A-CDK2 inhibits the transcritpional activity of E2F - which is then tagged with ubiquitin and is degraded
Cyclin E transcription promotes driving of
E2F
What is the purpose of the spindle checkpoint
If a chromosome has been misplaced the cell cycle will be paused allowing time for the stray chromosome to be captured
Progression to anaphase controlled by
The anaphase promoting complex
APC/C
Anaphase promoting complex
When is the APC/C active
When all of the chromosomes are aligned on the spindle
APC/C is a
Ubiquitin ligase
What does APC/C ubiquitinate
Securin - causing the degradation of securine
What is securin bound to
Separase
When does separase become active
When securin has been ubiquitinated and degraded - and separase has been released from its complex with securin
What is the role of separase once it become active
Breaks the links between the 2 sister chormatids
How does separase break the links between the 2 sister chromatids
Degrades cohesin
What is checked for at the G2/M transition
Integrity and fidelity of the DNA
DNA replication
DNA damage checkpoints are active when
G1, S, G2
What protein mediates cell cycle arrest when there is a double strand break
ATM
What protein mediates cell cycle arrest when there is a single stranded break
ATR
P53 is a
Tumour suppressor gene
What molecular function does P53 have
It is transcription
What genes are regulated by p53
Genes which mediated growth suppression, apoptosis and repair
What is the target gene of P53
P21(Cip1)
P21 is a
CKI
What does P21 inhibit
E-CDK2, A-CDK2, A-CDC2 and B-CDC2
Describe the normal mechanism of function of P53 in response to dNA damage
P53 detects DNA damage and activates transcription of target genes
- Cells halt at G1 checkpoint
- DNA repair activated
- Apoptosis
The underlying concept in P53 in response to DNA damage
Damaged DNA is not passed on to progeny
An effect of a mutation in P53 means that
P53 is unable to bidn to the DNA so can’t transcribe any of the genes
What is the outcome of mutation in p53
Damaged DNA is passed on
What is anoikis
Cell detachement induced apoptosis
What is the point of anokisis
A self defence stratergt that organsisms use to eliminate cells in inapproporiate locations
Myc protein governs
The deicsion of proliferation vs differentiation
More that 70% of human tumours overexpress
A member of the myc family
What are the members of the myc family
C/N/L myc
How many ways of myc inductions
3
Describe breifely the three ways of myc induction
Expression dirven by a normal promoter but gene is amplifed due to an abnormal copy number
Chromosomal translocation - puts myc under control of a foreign promoter
Pro-virus integration causes insertional mutagenesis
What type of molecule is myc
bHLH TF
Describe the strucutre of myc
basic DNA binding domain followed by an amino acid sequence forming an alpha helix, a loop and then a second helix
Myc forms …… which then becomes associated with gene promoters
Dimers
What is the effect of myc-max on proliferation
Promotes
What is the effect of myc-max on differentiation
Inhibits
What is the effect of mad-max on proliferation
Inhibition
What is the effect of mad-max on differnetiation
Promotes
Mad is expressed more highly in what cells
Differentiating cells
The higher levels of expression of Mad in differentiating cells means
Mad able to dispalce myc from its complex with Max
What does myc/max also regulate
The expression of key componenets of the celll cycle including C2 and CDK3
Also has an effect on E2F transcription facotrs
What is the effect of myc-miz1
Repression of CKI liberating E/CDK2 complexes from inhibitio n
What is the effeect of myc-miz1 on P27
Deg
Describe an experimental set up of the Myc oncoprotein
Myc expressed as a fusion protein with oestrogen receptor
Addition tamoxifen or oestrogen (ligand) drives myc translocation to the nucleus
What is the effect of adding tamoxifen to cells that are in G0 in the absence of any growth factors
tamoxifen addition induces re-entry back into G1 and S phase
The conclusion of the ‘powers of myc protein’
Myc is acting on its own and is able to lift all of the constraints on proliferation
What is TGF-B involved in
Growth Differentiation Apoptosis Homeostasis Other cellular functions
What receptor does a TGF-B ligand bind to
Binds to the type II receptor which then recruits and phosphorylates the type 1 receptor
Type 1 receptor phosphorylates
Receptor regulated SMADs
What can happen once the type I receptors has phsophrorylated the R-SMADs
Can then bind to the coSMAD - SMAD4
TGF-B strongly increases levels of …..
This causes ….
End result of this is that ….
Levels of P15
Inhibition of C-CDK4/6
Cells are unable to reach the R point
TGF-B weakly induced
Levels of P21
TGF-B uses ____________ to block progression through
2 CDK inhibitors (p15 and p21) to block progression through G1
TGF-B inhibits the expression of
Myc
Is TGF-B still able to inhibit the expression of myc in tumours
No
TGF-B _________ the activty of Myc
It also stops the binding of _______ to CKI promoters
Counteracts
Myc
What are two examples of how cancer cells inactivate TGF-B signalling
Mutations in SMAD4
Mutations in colon cancers
TGF-B inhibits cell cycle progression by controlling pRb function
How do cancer cells get aorund this
Deregulation of the pRB pathway
What are the mechanims of deregulation of the pRB pathway
Inactivation of Rb through mutations
Expression of very high levels of cyclin D1
Downregulation of INK4 proteins - inapproproate activation of pRb
Mutations in CDK4 gene - no longer able to bind the INK4 familty
P53 is a TF for genes
Regulating cell growth and apotosis
P53 is the most …
Freuqentyl mutated gene in human cancers
Where do mutations in p53 tend to occur
In the DNA binding domain
Control of p53 levels is through _____
Mdm2
Describe how p53 is controlled by Mdm2
Ubiquinated by Mdm2 and degraded by the proteaosome in the absence of stress
When does Mdm2 cause the degradation of p53
When there is no stress
What must happen when there is stress or damage
P53 must be protected from degradation
What is the mechanism for the protection of P53
Phos of P53 blocks Mdm2 binding and this is mediated by ATM/ATR/Chk2
What mediates all 3 of the DNA damage checkpoints
P53
Describe what must happen to P53 when there is stress of damage
P53 must be protected
Phos of P53 blocks Mdm2 binding
ATM phos Mdm2 –> inactivating Mdm2
Rapid increase in p53 levels
What is the effect of mitogenic and cell curvival signals
Induction of Mdm2
Phosphorylation induced activation of mdm2 (at a different residue
P53 is degraded in the proteasome
Supression of P53 levels
How does p53 cause cell cycle arrest
By upregualting P21 which inactivates cyclin-CDK complexes
