L3 - Evading Growth Supression Flashcards
What underpins all of the mechanism of growth supression
The cell cycle clock
Where do most growth supression mechanisms act
At the restriction (R) point
Where is the R point
At the end of G1 phase
This is where the cell must commit to division
Cells spend most of their tiime in what phase
G1
What is the most simple way to detect cells in S phase and mitosis
Cell imagine
What stains are used to determine if a cell is in S phase of M phase of the cell cycle
PCNA marker - marking sites of DNA replication - double staining with DNA
Stain for tubulin - reveals mitotic spindle - if their is one
What doesn’t cell imagine solve
The G1/G2 probelm
What is the difference between the cells genetic content in G1 and G2 phase
DNA has be replicated in S phase
So DNA in G2 phase has double the ammount of DNA compared to in G1 phase
How can we sort cells into those with n and 2n of DNA
Using a fluorescent marker - stains the DNA - ammount of staining will depend of how much DNA there is to stain
Then use fluorescent activated cell forting to sort
Describe how FACS is occuring
Measuring the amount of regular light that is being scattered - the amount of light that is being blocked is proportional to the size of the cell
Descibe how if we know how long it takes for cells to double we can work out how long cells spend it a phase
Measure the proportion of cells (0.xx) and multiply this by the the total length of time for the cycle
e.g. mammalian cells double ever 22 hours and at any one time 45% are in G1
Length of G1 = 0.45 x 22 = 10hours
Describe the three dimensions used in £D FACS analysis
Cell numbers
The relative ammount of DNA per cell
Rate of DNA synthesis
Describe how the rate of DNA synthesis is measured
Introduce a modified nucleotide which can be incorporated during S phase
Mimics T and incorportates over 30 mins
Can detect the modification using fluorescence
RATE OF INCORPORATION IS PROPORTIONAL TO THE RATE OF DNA SYNTHESIS
What is an advantage of 3D FACS
Allows us to distinguish between cells in Sphase arrest and G1 arrest
Allows us to identify detailed mechanisms of cell cycle arrest
When is G1/S cyclin high
High during G1 - drops at the start of S phase
When is S cyclin high
Builds up following the restriction phase falls following the entrance into M phase
When is M cyclin
M cyclin builds up during G2 phase and falls after the metaphase-anaphase transition
What is the idea behind synchronising cells
What is the technique to achieved this
Get them all in the same phase of the cell cycle
Mitotic shake off
Describe how mitotic shake off would be performed
When in interphase cells have a flattened morphology with a diffuse microtubule network
Mitotic cells rounded with a flattened morphology - THESE ARE VULNERABLE TO SHEAR FORCE
Knock the cells off with a shear force and collect all of the mitotic cells in a new dish
What is SLBP
Stabiliser of histone mRNA - made exclusively in S-phase
Replicator selection
The idea that the places where replication will start is determined early - leads to the formation of pre-replicative complexes
When are pre rec complexes made
In G1 phase without DNA polymerse
What does replicator selection ensure
That DNA replication from one place only happens once
Describe how repilicator selection only happens once
Cdt1 binds to the origins of replication and once replication begins this is proteolytically degraded
The action of Cdt1 ensure
Cell cycle directionality
What is geminin
Mops up any remaining Cdt1 to prevent rereplication
Expressed at late S/early G2
Describe the patterns in expression of Cdt1, SLBP and geminin
Cdt1 - expressed first then SLBP and then geminin
When is SLBP needed
During late G2
Histone H1 needs to be made to allow the reorganisation of chromosomes early in mitosis
Why is 4 colour fluourscence hard
Because need to have different excitation spectrum to absorption spectrum 4 times over the range of wavelengths for visible light
G1 marker
S phase marker
G2 marker
M markers
g1 Cdt1
S SLBP
G2 geminin
M histone H1
How can 4 tag fluorescence be used to monitor cell cycle progression
Can track the markers over time - compare to the colour indicators
What is the main statement that supports the existence of tumour supressor genes
Cancer phenotype is recessive
What experiments did Rao and Johnson perform
Formation of heterokaryotons - monkey and mouse cells
What is a heterokaryon
Cell with multiple nuceli
What was seen when a cancerous and normal cell were fused and injected into an immunocompromised mouse
No tumour was seen sicne the cancer phenotype is recessive
GENETIC RESCUE - absence of the TSGs recovered by the presence of the wildtype alleles
What is more likely gain or loss of function mutations
Loss
Evidence for and against the existence of tumour suppressor genes based on the likelyhood
YES - loss of function is more likely to occur than gain of function
NO - would need to lose both alleles - this is highly unlikely
Retinoblastoma
Morphology
Treatment
Whitening of the iris - can lead to blindness
Treatment involves the removal of the eye
Two forms of retinoblastoma
Sporadic and famililal
Sporadic is usually
Unilateral
Familial is almost always
Bilateral
Describe the incidence of non-retinal tumours in retinoblastoma patients
Increased
At 50 y/o
Bilateral patients have a 36% likelihood
Unilateral have a 5.7% likelihood
Describe Knudsens observations when ploting percentage of cases not yet diagnosed and age in months
For famililar - straight line
For unilateral - curve
What did the straight line for bilateral indicated
FIRST ORDER REACTION
Suggests that there is one thing involved - one thing needs to go wrong
What did the curve line for unilateral indicated
SECOND ORDER RATE Two things (two reactants) need to go wrong for cancer to occur
Describe the two hit hypothesis for familial Rb
Inherit the mutant Rb
Mutation then must occur in the paralogue
Describe the two hit hypothesis for sporadid Rb
Acquire the first Rb mutation
THEN have to also acquire a second mutation
Describe how this second mutation can occur so frequently
Mitotic recombination frequent between sister chromatids
Sgregation may then lead to daughter cells with two copies of the mutant gene
LOSS OF HETEROZYGOSITY
What chromosome is implicated in retinoblastoma
Chromsome 13
What is the technique to determine that loss of heterozygosity has occured
Zymography
Describe the technique of zymography
Using Desterase which has two forms - each isoform has a different molecular weight
Run a PAGE and incubate the substrate and it will generate a product which is fluorescent and can be viualused
What is seen when running zymography of a tumour tissue
People never have both of the isoforms in the tumour tissue
Heterozygosity is always lost first
What does the Rb protein interact with
E2F
What is E2F
What genes are under its control
Principle transcription factor
Drives transcription of all genes responsible for S phase
Describe E2f in the absence of mitogens
E2F binds Rb - E2F is blocked from activating its target genes
Describe E2F in the presence of mitogens
Mitogens turn on mRNA for cyclinD
Turns of Cdk4/6
Phosphorylates Rb to the hypo phosphorylated state
Cyclin E - Cdk2 then phosphorylates E2F to the hypephosphorylated state
Causes Rb to fall off E2F and the chromatin
Acetylation of S phase genes
E2F genes are now able to be transcribed
Describe the MAIN effect on E2F genes of mutant Rb
E2F target genes are ALWAYS on
What is the effects of mutant Rb being present
Upstream E2F signalling is bypassed and now no longer relevant
