L5 - Limitless Replicative Potential Flashcards
Why is C.elegans a useful model system
We know the lineage of all 959 cells
Cells appear to count …
The number of population doublings (generations)
What did Hayflick demonstrate?
That cells dont grow forever
Describe the graph whe looking at the number of population doublings
S shaped graph
Initailly (1) slow cell growth Then rapid (phase 2) cell growth
Cells then reach a point of senescence (this is known as the Hayflick limit) where cells NO LONGER DIVIDE
How many population doublings does it take to reach the Hayflick limit
Around 60
What can be seen if take cells from older donors regarding the doubling number
Doubling number tends to decline with age
What would be seen if you take cells that have doubled say 30 times and then freeze them …
What conclusions can be made from this
That cells will only go through another 30 divisions before they begin to senesce
In other words the cells remember where they are … and always bein to senesce at the same cumulative number of days
What gene can be used to overcome the senescence block
SY40 large T antigen
Describe how the large T gene could be introduced into cells in a way that it could be easily turned on
Transfect cells with a plasmid containing the large T gene
Use an inducible promotoer - e.g. glucocorticoid and then add dexamethasone
Give an example of a compund that would actvviate a glucocorticoid pormoter
Dexamethasone
Once dexa has been added how can you verify that transfection has occured
Add dex and detect large T through immunoblotting
Describe what happens to cells in the presence of dex
Cells grow as normal until the crisis point - many die but around 1 in 10 emerge and are now immortalised
Describe what happens to the cells if dex is taken away at any time
Cells senesce
The fact that cells senesce at any time if dex is withdrawn suggests what?
There must be a second block to immortal cell growth
Cance cells must become ______ for
Immortal for tumours to form
How many cells are required in a tumour for it to be fatal?
10^12
Why is the fact that 10^12 cells are needed for a tumour to be fatal strange?
This is ONLY 40 doublings and is well below the Hayflick limit
WHAT then is the point of senescnce??
What is the real story of how cells grow in a tumour
Alot of cell death - not true exponential cell growth
After 12 doublings there are around 5 cells not 2048
What are the reasons for the huge ammount of cell death within tumours
Deprivation of growth factors
Lack of vasculature
Deprivation of oxygen
What assay may be performed to detect cells underogiing apoptosis and their progression through apoptosis
Tunnel assay
Describe how the tunnel assay works
Enzyme that recognises chromosomal fragmentation - label with a flurophore
More fragmentation that occurs means the cells is further into apoptosis
Levels of what protein increases as the Hayflick limit is approached?
How can this be visualised
P16
P21
Western Blotting
Describe the actin cytoskeleton in normal cells and senescent cells
See a huge increase in the number of stress fibres in senescent cells
What is the effect of transfecting P16 into early human endometrial fibroblasts
Increase in stress fibres
Now has the morphology of a late cell
Describe the effect on senescence of the elimination of p53
Decrease in p53
How was p53 eliminated
Expose to a virus encoding a p53 siRNA
How was p53 knockdown confirmed
Using western blot
What were the results of p53 knockdown
On P21 and p16
On the ammount of cells going through apoptosis
Decrease in P16 p21
Decrease in the number of cells going through apoptosis
What is the function of P16
Binds to the catalytic of cdk4 and cdk6
Tweaks the regions to prvent ATP binding - so now no longer able to function as a kinase
Where does p16 act
At the restriction point
Where does p21 act
At the G1/S transition
What is the function of both Cdk4 and cdk6
So what occurs if they are inhibited by P16/21
Phosphorylation of Rb –> leads to the expression of E2F target genes - without this Rb not phosphorylated and E2F genes not transcribed
What is the action of P16 and P21 dependent on
What happens in cells when this critical component of the pathway is missing?
function of Rb
Where there is no Rb - senescence is not possible
What provides a basis for the ability of a cell to count its divisions
Teleomers
What is the function of telomeres
Prevent cell death from arising due to chromosomal end-to-end fusion
Describe how you could test the absence of chromosomes
Deletion of essential telomere protein TRF2
What is the results of a deletion of Trf2
Loss of telomeres
Chromosomes join together with multiple centromeres
Problems during segragation - breakage occurs at molecular weak points
End result is chromosomal end swapping
What are telomeres composed of
Hexomeric repeates of TTAGGG
Gives a G and C rich strand
What strand provides the single strand overhang
3’ G rich strand
How to telomers preserve chromosomal integrity
Looping able to occur - 3’ end tucked up into a loop
T-LOOP forms
How then are telomeres able to function as a clock
Their is leading and lagging strand synthesis
At the 5’ end - lagging strand, RNA primer doesn’t quite fit perfectly so at the end of every cycle the telomere will shorten
Describe how you would measure chromosomal end shortening
Extract chromosomal DNA
Restrict with multiple enzymes that do not target TTAGGG
Run DNA electrophoresis
Transfer to membrane
Label with DNA probe antisense to the telomere
Souther blotting - visuaise with AUDIORADIOOGRAPHY
What can be seen regarding the length of telomeres and time
Telomere length shortens as a function of time
What is responsible for the replication of telomeres
Telomerase
What is telomerase
A DNA polymerase of the reverse transcriptase class RNA --> DNA
What is unique about telomerase
It has its own template
What could be used to measure telomerase activity
TRAP assay
What can be said of the telomerase activity of an immortal cell
Has increased telomerase activity
What is hTERT
The catalytic subunit of telomerase reverse transcriptase
What may be avoided by expressing HTERT in cells
Crisis may be avoided
Describe how telomerase activity could be targeted for therapeutic benifit
Tumour cell growth can be blocked by interfering with telomerase function
If immortalisation via telomerase expression is important for tumorigenesis
Cancer cell proliferation might be blocked by interfering with telomerase activity
What correlation was seen following transfection of HTERT
Length of time following transfection to see an inhibition of cell growth is a function of the length of the teleomeres in cells
What cancer types can telomerase expression be used as a prognostic marker for
Neuroblastoma and Ewings Sarcoma
What is seen in cancers that have negative/low telomerase levels
Prognosis is quite good
Why s it the case that many neuroblastomas fail to transit to immortality
Early neuroblastomas have not yet gone through crisis
So … many neuroblastomas dont transit to immortality
