Tumour Suppressor Genes Ii

Front 1

How does oncogenic Ras stimulate cell proliferation?

Back 1

Oncogenic Ras short-cuts the need for an extracellular mitogenic signal to stimulate cell proliferation

Front 2

Oncogenic stress drives cell proliferation.

But oncogenic stress also results in the activation of what?

Back 2

CKIs

Front 3

Sustained E2F activation results in the up regulation of what? How?

Back 3

Front 4

Oncogene-dependent activation of p16 and p21 results in what?

Back 4

Front 5

What happens is Rb is lost?

Back 5

Sustained p53 activation promotes apoptosis

Front 6

Describe the inhibition of Rb and p53 tumour suppressors by DNA tumour viruses

Back 6

The HPV E7 protein, which is expressed early in the viral life-cycle, binds the 'pocket' of Rb, displacing it from E2F. E2F is therefore freed from Rb-dependent inhibition.

This activates the host cell's replicative machinery which the virus hijacks for its own propagation.

Additionally, the HPV E6 protein binds and inhibits p53, preventing apoptosis. This results in uncontrolled cell proliferation and survival.

The E7 and E6 viral proteins also promote the ubiquitination and degradation of Rb and p53.

Front 7

In the absence of Rb (targetted E7 expression), how is lens development affected?

Back 7

Lens fibre cells fail to withdraw from the cell cycle and fail to differentiate

Disorganised proliferation is counterbalanced by a high rate of apoptosis, consequently no tumours arise

Consequently no tumours arise

Front 8

How is lens development affected in the absence of both Rb and p53? (targeted expression of both E7 and E6).

Back 8

Lens fibre cells fail to withdraw from the cell cycle and fail to differentiate

However, apoptosis does not occur and lens cell division is unchecked.

Consequently, lens tumours develop.

This shows that the apoptosis response to Rb loss is p53-dependent.

Front 9

Is loss of RB1 the only genetic change needed for Retinoblastoma?

Back 9

It is now known that inactivation of the p53 tumour suppressor pathway also occurs in retinoblastoma

This would be predicted from experiments that show that Rb loss alone results in p53-dependent apoptosis

Tumourrigenesis is a multi-step process involving many genetic changes

Front 10

p53 is a 53kDa _____________ _________ encoded by the ______ gene

Back 10

transcription factor

Front 11

p53 monomers associate to form a _________ _______________, which is recruited to p53-specific response elements in the ___________ of target genes

Back 11

174 kDa tetramer

promoter

Front 12

More than ____% of human tumours contain a mutation or deletion of the p53 gene

Back 12

50

Front 13

Chromosome __ long arm deletions are often accompanied by ___________ in the remaining TP53 allele

Back 13

17

mutations

Front 14

Many tumours lack p53 expression all together.

WT p53 actively ____________ oncogene-mediated cellular transformation

Back 14

suppresses

Front 15

p53 is a key node in signalling networks involved in sensing cellular stress

What activates p53?

What are some of the responses p53 can generate?

Back 15

Front 16

The response that p53 generates is dependent on the stress it encounter. Give an example.

Back 16

If a cell is exposed to metabolic stress (e.g if energy/resources are scarce), you don't want the cell to go through the cell cycle because it probably doesn't have enough energy to replicate its genome. It makes more sense to transiently stop proliferation and wait for nutrients to become plentiful again, because going through the cell cycle. Under these cases, you might expect p53 to induce transient cell cycle arrest. It would be ridiculous for p53 to induce apoptosis whenever resources were depleted. In contrast, if the cell suffers massive DNA damage, i is a threat to the organism, so p53 induces apoptosis.

Front 17

The biological functions of p53 are mostly due to the activities of p53-regulated gene products.

What are some of these gene products?

Back 17

Front 18

Describe the p53 life cycle in healthy cells, including the role of MDM2

Back 18

In normal cells, the p53 gene should always be active, so p53 monomers are produced, which form a tetramer. The tetramer binds to the MDM2 promoter, and activate transcription of the MDM2 gene. MDM2 mRNA is exported to the cytoplasm, where it is translated to MDM2 protein. Act is present in healthy cells, and phosphorylates MDM2 in the cytoplasm, promoting the nuclear import of MDM2.

MDM2 is a ubiquitin ligase. MDM2 binds to and ubiquinates p53, and so p53 is targeted for degradation.

Front 19

p53 is activated by disrupting the ability of MDM2 to bind to p53. Why is this a better method of activating p53 vs increased transcription?

Back 19

Because it takes time for a stress event to induce the transcription and then translation of MDM2.

This mechanism mans a rapid response is induced in response to stress.

Front 20

In normal cells, what are p53 levels like and why?

Back 20

Low

Due to the short half life of the protein (6-20 mins)

Front 21

How does the p53 negative feedback response affect the levels of MDM2 and p53?

Back 21

Front 22

In normal cells p53 levels are low due to the short half-life of the protein (6- 20 min). This is because p53 is targeted by the E3-ubiquitin ligase MDM2.

Ubiquitination of p53 by MDM2 results in its nuclear _______ and ____________-mediated degradation.

However, MDM2 is a major _______________ target of p53 and as p53 levelsdiminish so, therefore, do those of MDM2.

Back 22

export

proteasome

transcriptional

Front 23

Arf only gets upregulated when there is excessive signalling from _____

Back 23

E2F

Front 24

Oncogene activation results in increased transcription of Arf. How does this up regulate p53?

Back 24

When Arf levels accumulate in response to oncogenic stress, Arf binds to MDM2, which relocalises it to the nucleolus (i.e it is physically speared from p53 so can't bind to it). This results in rapid accumulation of p53.

Front 25

What does the CDKN2A gene encode?

Back 25

Both the p16 and Arf tumour suppressors

Front 26

The CDKN2A gene encodes both the p16 and Arf tumour suppressors.

How come?

Back 26

p16INK4a and Arf transcription are initiated from different promoters and their respective mRNAsuse different exon combinations

Arf protein is encoded by an alternate reading frame (Arf) to that of p16INK4a

Consequently, although closely related at the genetic level, these two tumour suppressors have completely unrelated amino acid sequences and protein structures

However, they play related and extremely important roles in the Rb pathway

Front 27

Germline mutations in the CDKN2A gene are observed in 20–40% cases of _______ ____________

Back 27

familial melanoma

Rare cases in which CDK4 mutations have been associated with melanoma reveal a functional change that disrupts p16INK4a binding

Front 28

Germline mutations in the CDKN2A gene are observed in 20-40% of cases of familial melanoma.

Rare cases in which CDK4 mutations have been associated with melanoma, reveal a functional change which disrupts what?

Back 28

p16 bind

Front 29

Oncogenes such as myc and Ras promote the hyperactivation ofmitogenic signalling pathways (e.g. by up regulating ________expression)resulting in the __________________ and inhibition of Rb.

This in turndrives _______________ gene transcription.

Back 29

cyclin D

hyperphosphorylation

E2F-mediated

Front 30

Deregulated E2F activity promotes the transcriptional activation of the _____ protein.

Once expressed, _____ is recruited to the nucleus where it binds andsequesters MDM2 to the ____________, preventing its interaction with p53

Back 30

Arf (x2)

nucleolus

Front 31

DNA damage results in phosphorylation dependent ______________ of p53 and _______________ of MDM2

Back 31

stabilisation

inhibition

Front 32

DNA damage results in phosphorylation dependent stabilisation of p53 and inhibition of MDM2.

How?

Back 32

DNA damage is detected by ‘sensor’ proteins which recognise eithersingle-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) breaks.

These sensors then recruit protein kinases that function as transducers ofthe DNA damage signal, phosphorylating downstream effector proteinsincluding p53 and MDM2.

dsDNA breaks are recognised by the MRN sensorcomplex which recruits the protein kinase ATM.

ATM then phosphorylates p53 either directly or indirectly via theactivation of the checkpoint kinases (Chk1 and Chk2).

DNA-PK (DNA-dependent protein kinase) is also recruited to dsDNA breaks.

ssDNA breaks are recognised by the sensor protein RPA (replication proteinA) which then recruits the protein kinase ATR .

ATR phosphorylates the Chk1/2 kinases, thereby promotingthe downstream phosphorylation of p53.

ATR, ATM and DNA-PK phosphorylate Ser15 of p53, while Chk1/2phosphorylate Ser20.

These residues lie within the N-terminal transactivationdomain of p53 and regulate MDM2 binding. Ser15/Ser20 phosphorylation ofp53 inhibits MDM2 binding, resulting in p53 stabilization and accumulation.

In addition to targeting p53, the Chk1/2 kinases also phosphorylate MDM2(at a residue district from that targeted by Akt), reducing its affinity for p53.

Front 33

Rising p53 levels induce cell cycle arrest through the transcriptional activation of ____, a CKI that targets and inhibits the _______________ complex, the activity of which is requires for G1/S transition.

Back 33

p21

Cyclin E/Cdk2

Front 34

What does Akt phosphorylation of MDM2 promote?

What does Chk1/2 phosphorylation of MDM2 promote?

Back 34

Nuclear entry and p53 association

p53 dissociation

Front 35

What happened one the DNA damage has been repaired?

Back 35

The DNA damage signals dissipate, p53 (and therefore p21 levels) declines, removing the barrier to cell cycle progression

Front 36

What happens if DNA damage is so extensive that it is beyond repair?In

Back 36

p53 can induce apoptosis, preventing the transmission of potentially dangerous genetic error to progeny cells

Front 37

More than 50% of human tumours contain a mutation or deletion of the TP53 gene.

The majority of these mutations are what kind of mutation?

Back 37

Missense mutations that impair p53-DNA binding

Front 38

In tumours that retain a normal copy of the p53 gene, other elements of the p53 pathway are often defective.

Give some examples (3).

Back 38

1. MDM2 or MDM4 over expression (e.g retinoblastoma-MDM2/4 levels exceed the sequestering capacity of Arf)

2. Inactivation of DNA-damage kinases (e.g germline mutations in Chk2 have been found in some Li Fraumeni-like families rather than p53 mutations)

3. Defective transcriptional regulation of p53 (e.g some breast tumours have low levels of p53 due to suppression of HOXA5, a transcription factor involved in p53 expression)

Front 39

While some tumours exhibit complete loss of p53 expression or carry p53 mutations that result in loss of protein function, others express p53 mutants that antagonise the function of the wild-type protein.

What kind of mutations in p53 are these?

Back 39

Dominant-negative p53 mutations

Gain-of-function p53 mutations

Front 40

How do dominant negative p53 mutations disrupt normal p53 function?

Back 40

These mutants interfere with the function of normal p53 by forming inactive tetramers

Therefore, a similar result is obtained from mutating one allele as from deleting both

Front 41

Other p53 mutations have been found to have a gain-of-function phenotype.

What observations resulted in the identification of p53 mutants which exhibit pro-oncogenic functions not usually associated with the wild-type protein?

Back 41

Breast and colon cancer patients whose tumours carry certain missense p53 mutations tend to have a poorer prognosis than patients with p53 null tumours

Expression of certain p53 mutants in ells lacking normal p53 enhances the cell's tumorigenic potential

Front 42

What are 3 kinds of gain of function mutations in p53?

Back 42

Altered target gene specificity (the inappropriate transcriptional activation of genes associated with cell growth/survival ,e.g E2F, NFkB. These mutations likely alter the spectrum of gene promoters recognised by p53)

A switch from transcriptional repression to activation (the transcriptional activation of genes usually suppressed by p53 function, e.g c-myc)

Production of chemoresistance (up regulation of genes associated with cancer chemoresistance-a p53 mutant was shown to stimulate the multi drug resistance (MDR1) gene whereas WT p53 represses its expression)

Selective loss of specific p53 transcriptionaltargets• The p53 mutant (R213Q) is defective in its ability to up-regulate cell cycle arrest proteins and pro-apoptotic factors, but is highly effective at activating MDM2 (Such a mutant is likely to compromise the function of the WT protein)

Direct inhibition of signalling pathways throughaltered protein-protein interactions-The p53 mutant (R248W) binds and inhibits the MRN complex, preventing its recruitment toDNA double-stranded breaks. This inhibits ATMfunction, thereby providing a mechanism forincreased genomic instability.

Front 43

What determines the nature and severity of the p53 dependent response?

Back 43

1. Amplitude and duration of p53 activation

2. The different covalent modifications of p53 (p53 can be modified through ubiquitination, phosphorylation, acetylation and methylation)

Front 44

The amplitude and duration of p53 activation dictates the final outcome of a p53 dependent response.

Explain this in a little more detail.

Back 44

If p53 is induced for a long time at high levels, perhaps it is more likely to induce transcription from promoters which are less sensitive to p53 (e.g pro-apoptotic genes), vs promoters that are sensitive to p53 (e.g MDM2)

Front 45

How does the phosphorylation status of p53 affect its activity?

Back 45

Low levels of p53 phosphorylation are sufficient to displace MDM2, while higher levels promote the recruitment of transcriptional co-activators.

Because the affinity of p53 for the promoters of target genes varies (high: MDM2 &21 vs low apoptotic genes), higher levels of phosphorylation may be required to promote transcription of less sensitive genes