L5 - Limitless Replicative Potential Flashcards
(55 cards)
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The fact that cells senesce at any time if dex is withdrawn suggests what?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
