5. p16 and senescence Flashcards
what has p16 been shown to interact with? and how was this shown?
cdk4 and cdk6
>this was shown in a Yeast-2-hyrbid screen using a histidine reporter
>the then proved this interaction in mammal cells using immunoprecipitation
how often is p16 mutated in cancer cells lines? what sort of mutations are seen and what does this imply?
it is mutated very frequently in cancer cells lines
> these mutations are mostly deletions
> this implies that a tumour suppresser has been knocked out
describe the structure of p16
4 ~ 33-residue Ankyrin repeats
these are helix turn helix motifs that interact and stabilised each other
what part of p16 provides binding specificity?
the loops provide binding specificity of protein-protein interactions
what is seen when p16 is added to the cdk cyclin complex that it binds?
p16 kicks cyclin D off cdks4
> there is reciprocal binding of p16 and cyclin D to cdk4
what might an inactivating mutation of p16 found in tumours result in?
it no longer being able to interact with cdk4 and cdk6
a kinase assay was done titrating p16 with cdk4/6, what was observed? and what is observed with mutant p16?
high levels of p16 inactivate kinase
mutant p16 does not bind cdk4/6 and so kinase activity remains the same/is uninhibited
describe how p16 binds cdks
> p16 binds the opposite side of cdk4/6 to cyclins
how does the binding of p16 affect cdks
p16 affects cyclin binding site in an allosteric way and does not directly interact with the binding surface
describe what happens to the structure of cdks when p16 binds
> the N lobe of the kinase rotates by 15 degrees, this misaligns ATP binding site
15 degrees twist in alpha helix which is important for cyclin binding is displaced so that cyclin can no longer bind
what two things does the binding of p16 to cdks inhibit? and how does p27 differ from this?
cdk4/6 binding to ATP and cyclin D
> p27 sterically blocks the ATP binding site and the substrate recruitment by the cyclin
what can be deduced from the amount of DNA in a cell?
the stage in the cell cycle
what phases of the cell cycle will cells have double as much DNA?
G2 and M phase
what can affect the proportion of cells in different cell cycle phases when p16 is added?
whether Rb is knocked out
when p16 is added to Rb WT cells, what is seen in flow cytometry experiments? and how is this different to mutant p16?
there is a large peak in the G1 phase with no cells in G1 and M phase
> populations of cells in different stages of the cell cycle are similar to that of the control with no p16
what is seen when p16 is added to Rb null cells? and what does this indicate?
the addition of WT and mutant p16 has no affect on what stage of the cell cycle Rb null cells are in
what is the only essential substrate of cdk4 to allow cell cycle progression? and why is this?
Rb - along with additional phosphorylation this allows the release of E2F
why does p16 have no affect on Rb null cells?
E2F is already free and active therefore inhibiting cdk4 has no affect
how can p16 indirectly have an affect on cdk2?
> there is always a basal expression levels of p27 which binds all cdks/cyclins
when p16 is added it displaces p27 from cdk4
this frees up p27 to bind other cdk/cyclins and so increases inhibition of cdk2 which reduces number of cells in S phase
what is normally expressed in high levels in Rb null cells, and what affect would adding more of this have?
p16
adding more would have little effect as there are no cyclinD/cdk complexes remaining
what happens when p16 is over expressed in Rb positive cells?
it causes G1 cell cycle arrest
how common are p16 mutations in primary tumour material? and comment on why this might be different from cell lines and how this might then compare to cell lines?
it is mutated less common than in cell lines
>p16 can also be inactivated by promoter methylation - this inactivates transcription
>this would still not equate to the number of mutations seen in cells lines
when DNA is methylated, what is made and what enzyme catalyses this reaction? and what do this do?
5-methyl-cycteine
DNA methyl transferases
> generally this represses transcription
what can be coupled with methylation to further silence a gene?
histone methylation by HDACs to compact chromatin
what can be seen when doing a restriction digest with enzymes sensitive to DNA methylation at p16 locus of carcinoma cells?
some cells have strong bands indicating methylation which is not seen in control cells
> this indicated the promoter is supressed in some cancer cells
what are the four other family members of the p16 family? comment on their structure and function
p15, p16, p18 and p19
> they have Ankyrin repeat loops that bind to cdk4 and cdk6
which two family members of p16 are involved in wide spread cell cycle regulation and what are the other two used for?
p15 and p16
p18 and p19 are involved in specific cell type proliferation and inhibition during development, and so are not associated as much with cancer
what do double KO p16 mice have a predisposition to?
tumours
why is there a difference in the number of time p16 is mutated in cells line and primary tumours? and why is this?
loss of p16 is required to establish a cells line
> this is because p16 is known to impose senescence
> when culturing cells you are selecting for cells are proliferating i.e. have lost p16
what is senescence?
the irreversible withdrawal from the cell cycle
what happens when p16 is expressed in high levels?
cells prematurely stop proliferating and enter senescence
senescence is a natural process, when does it occur?
it occurs when a cell has gone through too many division cycles
what process can be seen when cells are transfected with oncogenic ras? and why does this occur?
oncogene induced senescence
> activated Ras switches on p16 expression
what is a primary line of defence against oncogenes that can effectively stops tumorigenesis occurring?
p16
name the two types of senescence
stress-induced senescence
replicative senescence
what can cause stress induced senescence? (3)
> oncogene activation
DNA damage
oxidative stress
what happens to telomeres as a cell divides? and what affect might this have? and why is this through to happen?
they shorten
> when telomeres have reached a minimal length this triggers a DNA damaged response
> this is thought to be a fail-safe mechanism to stop cells from proliferating forever, accumulate mutations and lead to cancer
what do telomeres at the end of chromosomes act as? (2)
> protection
>a clock to monitor to monitor how many cell cycles a cell has been through
describe how chromosomes are replicated in S phase and how this leads to shortened telomeres
> RNA primer, synthesised by DNA primase, binds somewhere near the end of the chromosome (this is largely random where it binds)
this means that 3’ end of the chromosome will not be replicated
this acts as a starting point for 5’ to 3’ DNA synthesis by DNA polymerase
RNA primer is removed
how many bases are lost each replication cycle from the end of telomeres?
50-200bps
what are telomeres?
there are TTAGGG repeats structures that cap chromosomes
how do mice telomeres compare to ours?
they have more repeats that us
how many telomere repeats do humans have and how many bases does this equate to? and what does this mean?
500-4000 double stranded repeats
> 5-25kb
> lots of repeats can be lost without affecting gene coding regions
what is telomerase?
a ribonucleoprotein complex that elongates telomeres
what occurs when mice lack a vital component of telomerase (mTR)?
they are unable to replenish their telomeres and so mice look older than WT, this is true both physically and at the tissue level
name a rapidly dividing tissue and what is seen when mice lack a vital component of telomerase (mTR)?
> seminal vesicle that produces sperm
the tissue is broken down as rapidly dividing cells have gone through more cells divisions that less proliferative tissue
no sperm is produced by these mice
what happens when mTR is KO in Arabidopsis?
normally they can through multiple rounds of flowering, seed production and flower growth. when this is KO the plants age, lose rigour and morphology.
why is telomere length not an issue for most cells in our body? and what type of cells does this matter to?
they are post-mitotic
> stem cells and germ cells
describe telomeres in germ cells, explaining why this need to happen
> telomere length in germ cells never changes
> DNA has been passed down through generations and so these cells need to be able to proliferate indefinitely
describe the telomeres in stem cells
the telomere length in stem cells declines a little but not too much
telomerase expression is low or absent in what tissue?
most somatic tissue
where is telomerase expressed?
lymphocytes, germs cells and tissue stem cells
what type of cells do not go into replicative senescence? and why is this?
and how could this be therapeutically targeted?
cancer cells
>they either maintain their telomere length or increase them
>preventing telomere lengthening would eventually lead to replicative senescence
describe ciliated protozoa
> single cells with lots of cilia
40 million chromosomes
cannot undergo replicative senescence
they were used as the original source for isolating telomerase
what is the catalytic subunit of telomerase called and what is associated with it?
hTERT
an RNA component is associated with it
describe how telomerase functions
> telomerase RNA hybridises to end of chromosome
telomerase catalyses addition of DNA bases to end of chromosome using RNA as a template
this process is done repeatedly
what does increasing the length of the end of the chromosome mean?
this provides a greater chance for when you add in the RNA primer that this will land closer to the end of the chromosome
telomerase uses RNA template to make DNA, what other enzyme does this? and what relevance does this have to therapies?
reverse transcriptase
>telomerase is the only reverse transcriptase encoded in our genome and so inhibitors will be specific
what is telomere length a balance between?
> telomerase activity
>number of mitoses
germ cells and stem cells express tHERT, what other cells express this?
cancer cells
name one way to immortalise cells
add the genes that encodes for telomerase
increasing concentrations of what can block tumour progression? what is odd about this? and what does this imply?
small molecule inhibitor of telomerase
> they do this over a short period of time (e.g. 3-9 days) which is not enough time to trigger replicative senescence
>when you inhibiting telomerase, cells sense they be able to lengthen their telomeres indefinably in the future and so don’t divide
there is a link between cell cycle blockage and what? is this link known?
telomerase inhibition
how this is linked is not known
what else can be used to inhibit telomerase activity?
> anti-retrovirus drugs
>AZT is an analogue of thymidine
what happens when AZT is incorporated into DNA?
you cannot add subsequent bases
what is a side effect of telomerase inhibition? and what implication does this have on the treatment, and why is this a problem?
> germ cells are killed - infertility
stem cells are depleted - unable to replenish tissue
cannot give this drug to patients for a long time
need to be given for a long time as in short periods cells are not killed, they just stop proliferating
what percentage of tumour cells is telomerase activity sensed in? and what does this suggest?
85-90%
this suggests that 10% of tumours don’t have telomerase but can still proliferate indefinitely
what happens when telomerase activity is KO in fission yeast?
they circularise their chromosomes so that telomeres aren’t lost
what does ALT stand for?
alternative lengthening of telomeres
what does the ALT pathway do?
uses homologous recombination to maintain telomere length in telomerase null cancer cells
how does the ALT pathway work?
> if you have chromosome with a long telomere and one with a short telomere
inter-telomeric strand invasion where short telomere uses long telomere strand as a template to extend one of its own strand
it can use this strand as a template to synthesize the second strand
what does the ALT pathway lead to?
a net gain of telomere length
what does the ALT pathway rely on and why does this make it a hard therapeutic target?
> relies on many of the enzymes which are involved in standard DNA repair
inhibiting them will lead to accumulation of mutations and cancer
what does inhibiting telomerase often activate?
ALT pathway
