Lecture 3. p53 & ARF Flashcards
What is p53?
P53 is a txn factor - N-terminal txn actn domain, central DNA-binding domain, & C-terminal tetramerization & regulatory domains
Binds DNA as a tetramer, formed by a pair of dimers
Target genes include cdk inhibitor p21 (to arrest cell cycle) & death gene Bax (to induce apoptosis)
Txn actn domain stimulates txn by binding & recruiting TFIID & HATs (e.g. p300)
What is MDM2?
a key target gene of p53 encodes MDM2, a ubiquitin ligase that controls p53 level by polyubiquitination, causing proteasomal degrdn
prevents excessive accumulation of p53 & allows return to basal p53 levels once inducing stimuli are removed - negative feedback loop
MDM2 is essential because p53 can activate apoptosis - MDM2 knockout mice die in utero due to p53-induced cell death
Mutant p53 accumulates to much higher levels than WT p53 because MDM2 is not induced
Explain why MDM2 is a target for anti-cancer drugs
cancers with WT p53 sometimes overexpress MDM2 – e.g. many sarcomas
Nutlin drugs were developed to prevent p53 degradn by MDM2
MDM2 binds key residues in txn activation domain of p53
crystal structure of a p53 peptide bound to MDM2 allowed structure-based drug design
Several nutlin derivatives are in clinical trials
Phase I trials for various cancer types found increased p53 & p21 expression, confirming that drug reached its target
However, patients suffered adverse side effects, esp. nausea & haematological toxicity
Complete remission achieved for some patients with acute myeloid leukaemia (AML)
Second generation nutlin derivatives show better efficacy & can be used at lower concentrations with fewer side effects
Describe how p53 is regulated by post-translational modifications
In addition to polyubiquitination which occurs at range of lysine residues at p53 c terminus the regulatory region is subject to other modifications
Multiple serine/threonine residues undergo phosphorylation at both termini
Different modifications carried out by different modifying enzymes in response to different types of condition and can in turn affect p53 in a number of ways providing a great deal of regulatory flexibility
Different responses, Different stimuli, all from same protein
Describe how kinases regulate p53
ATM kinase example of post-translational modification activated by dsDNA breaks
Under these circumstances phosphorylates p53 on Ser 15 close to where p53/mdm2 interaction occurs – its phosphorylation blocks interaction
This stabilises p53 allowing it to accumulate and elicit a response to the DNA damage – be that cell cycle arrest and DNA repair or apoptosis
Other kinases phosphorylate other residues in response to other types of stimulus – nature of response varied, depending on stimulus and cell type
Explain how multiple stresses activate p53
p53 probably evolved as a coordinator of stress responses
different stresses trigger different types of response, in terms of PTM & target genes – extremely context-dependent
PTMs afford flexibility & allow responses to be tailored to stimulus
best studied is response to DNA damage, but is this the most important?
apoptosis in response to DNA damage may not be rqrd for tumour suppression by p53, at least in mouse models
What is ARF?
ARF is one of 3 tumour suppressor genes in a 35 kb locus that is often deleted in tumours e.g. melanoma
ARF & INK4a genes share the same 2nd & 3rd exons, but these are translated in Alternative Reading Frames to give polypeptides with no sequence similarity
ARF & INK4a have separate promoters & 1st exons
ARF protein binds MDM2, inhibits its ubiquitin ligase activity & sequesters it in the nucleolus
INK4a & INK4b genes encode p16 & p15 inhibitors of cyclin D-dependent cdk4 & cdk6
ARF promoter has E2F sites & is induced by oncogenic signals e.g. sustained actn of E2F after RB inactivation
ARF triggers a p53 response
ARF also has p53-independent functions, including inhibition of ribosome biogenesis
What are focus formation and soft agar assays?
Simple assays to measure oncogenic transformation using cultured cells
Untransformed cells normally grow as a monolayer in culture & stop proliferating when they touch each other (termed confluence) due to “contact inhibition”
Transformed cells have lost contact inhibition (Hallmark: Evading growth suppressors) & form foci where cells continue to proliferate & pile on top of each other
Whereas focus formation assay grows cells on plastic, soft agar assay grows cells on top of a layer of agar
Untransformed cells will not proliferate without solid support – provided by extracellular matrix in vivo or by plastic dish in vitro
transformed cells are “anchorage independent” & form colonies in soft agar
Describe how tumorigenicity is tested in vivo
assays using cultured cells only measure certain aspects of oncogenic transformation e.g. proliferation, contact inhibition, anchorage-dependence
Xenograft assays are used to test tumourigenicity of human cells in vivo
Cells are injected under skin in flanks of immunodeficient mice
Immunodeficiency is necessary to prevent immune rejection
Tumour growth is measured over time using calipers
Mice must be culled when they show signs of suffering
genetically engineered mouse models are much more physiologically realistic, but sometimes fail to mimic human disease
E.g. humans who inherit a mutant RB allele develop retinoblastoma, but RB+/- mice develop thyroid cancers & never retinoblastoma
important to be aware that all models have limitations
How does p53 suppress tumours?
Despite decades of intense research, how p53 suppresses tumour formation is still hotly debated – probably depends on context
best characterized p53 responses are cell cycle arrest & induction of apoptosis
however, in knock-in mice, an artificial p53 mutant unable to induce apoptosis can still suppress tumour formation
other artificial p53 mutants can induce cell cycle arrest & apoptosis, but do not suppress tumorigenesis
Certain mutns in txn actn domain inactivate p53 as a tumour suppressor but still allow it to induce cell cycle arrest & apoptosis
Oncogenic stress can cause p53 to induce ARF-dependent senescence (stable cell cycle exit) that correlates with tumour suppression in some contexts
What is cellular senescence?
Senescence is a failsafe mechanism to prevent proliferation of cells at risk of oncogenic transformation – e.g. those with activated oncogenes
involves induction of p53 via ARF & activation of RB via p16INK4a
protection is compromised if cells lose ARF or p53 and p16 or RB - cancer can progress
Senescence is form of stable cell cycle exit
If you take a normal cell and induce mutant Ras you don’t get proliferation you see induction of p53 and its target p21 and induction of p16, inhibitor of cyclin D dependent kinases – leading to dephosphorylation and activation of Rb
Cells senesce
How can senescence be induced?
Senescence can be induced by radiotherapies & chemotherapies used to treat cancer – induce p53
Nutlin drug that induces p53 by blocking its interaction with Mdm2 also induces senescence
Senescence is assayed by staining for senescence-associated b-galactosidase (SA b-gal), a marker expressed in senescent cells
