L8 Flashcards
(41 cards)
Describe the discovery of p53
- 53 kDa protein that interacted with
the viral SV40 T-antigen
- 3T3 mouse fibroblast Ccell lysates incubated with N or T and then immunoprecipitated with:
N= normal hamster serum
T= hamster antiserum reactive with SV40-transformed cells - Also found to be present in non-SV40 transformed cells (F9; mouse embryonal
carcinoma cells) and in mouse cells transformed by exposure to a chemical
carcinogen - led to the conclusion that
p53 was of CELLULAR ORIGIN (ENDOGENOUS protein)
Is p53 oncogene? Describe what happens in each scenario
1) ras and p53 deletion mutant
2) ras + p53 val-135 point mutant
3) ras + wild-type p53
1) ras and p53 deletion mutant
- Deletion: supports transformation
2) ras + p53 val-135 point mutant
- 1) Initial observation: Co-transfection with ras results in transformation. Is p53 an oncogene?
3) ras + wild-type p53
- P53 suppresses transformation
Mutant alleles of p53 (TP53) are frequent in human tumors. What % of ovary tumors have a TP53 mutation?
50%
Describe graph on slide 7
Kaplan-Meier survival analysis
- Shows % survival of different p53 genotypes as a function of age (days). Individuals that are homozygous for wildtype p53 have the highest survival, then those who are heterozygous (+/-), and individuals with both mutant copies of p53 have the lowest % survival (development of sarcomas and leukemias)
Majority of mutant p53 alleles are _____ mutations. Most of them affect the area of the DNA fxning in what?
- Missense (a point mutation in which a single nucleotide change results in a codon that codes for a diff AA)
- Most mutations affect DNA CONTACT or PROTEIN CONFORMATION
Majority of mutant APC, ATM, BRCA1 alleles are _____ mutations
frameshift (deletion or insertion in a DNA sequence that shifts the way the sequence is read)
Describe the structure of p53.
Homotetrameric transcription factor
In a cell that is heterozygous for p53, ____ of the subunits may lack fully normal function
15/16
What is the effect of a mutant p53 allele?
The mutant allele has a dominant negative effect (MORE severe than a deletion allele)
- altered gene product acts antagonistically to the wild-type allele
How do somatic vs. germline TP53 mutations differ? How are they similar?
- SOMATIC TP53 mutations contribute to SPORADIC cancer
- GERMLINE TP53 mutations cause a rare cancer predisposition
called Li-Fraumeni Syndrome - BOTH somatic and germline mutations are usually followed by LOH during tumour progression
Many mutant p53 forms lose tumor suppressor activity and acquire _______ activities and they also gain new ______. Example of oncogenic properties?
- Many mutant p53 forms lose tumor suppressor activity and acquire DOMINANT-NEGATIVE activities and they also gain new ONCOGENIC properties e.g. when mutant p53 is transfected into p53 -/- null cells, the result was ENHANCED ABILITY to form TUMOURS
What are the 5 oncogenic properties (tumour initiation and progression) of mutant p53? What are their underlying mechanisms?
- Proliferation and survival
- Differentiation block
- Drug resistance and inhibition of apoptosis
- Genetic instability
- Angiogenesis, invasino, and metastasis
- Inactivation of wild-type p53
- Inactivation of p63 and p73
- Interaction w/TFs (e.g. VDR)
- Structure specific DNA binding
- NF-kb pathway activation
- Inhibition of autophagy
- Cooperation w/Ras in transformation
- ATM pathway inactivation
- Topoisomerase I activation
What are the 3 classes of p53 mutations?
- LOF (loss of fxn)
- Transcriptional regulation of genes that mediate growth-suppression, apoptosis, DNA repair etc. - DN (dominant neg)
- GOF (gain of fxn)
- transcriptional regulation of genes that mediate proliferation, drug-resistance, survival, and metastasis etc.
Draw a diagram connecting the 3 classes of p53 mutations
Slide 13
Which property of p53 is selected for during cancer development?
N/A
What are 6 stress signals cause a rapid increase in p53 protein levels? In response to these signals, what are the possible actions of p53?
p53 receives signals from an array of surveillance systems
- Lack of nucleotides
- UV radiation
- Ionizing radiation
- Oncogene signaling
- Hypoxia
- Blockage of transcription
Lead to increased p53 lvls. p53 can either fxn in
- cell cycle arrest
- > can lead to SENESCENCE (irreversible) or RETURN to PROLIFERATION (reversible) - DNA repair
- block of angiogenesis
- apoptosis
Nickname of p53
Master guardian
Using a diagram show the pathway of diff stress signals on p53 and its action
Slide 14
Describe the control of p53 levels by Mdm2, provide a diagram
Slide 15
Mdm2 = mouse double minutest
- In cases of cell stress, p53 activate downstream transcriptional targets inc Mdm2. When Mdm2 is transcribed and translated, its protein will bind p53. Mdm2 is a ubiquitin ligase, thus acts to ubiquitinate p53 and cause its degradation. This forms a NEG FEEDBACK loop; high lvls of p53 leads to transcription of p53 regulator, which binds p53 and cause its degradation. Thus half-life of p53 is very rapid ~20 mins.
What events lead to p53 STABILIZATION and DEGRADATION
Stabilization of p53:
- DNA damage sensing kinases such as ATM and ATR act via Chk2
and phosphorylate p53 (which prevents Mdm2 binding) and also phosphorylate Mdm2 itself (on N terminus) so it cannot associate with p53.
Degradation of p53:
- Via Mdm2
- Suvival signals act through AP1 and Ets TFs to collaborate with p53 to promote Mdm2 transcription and translation
- these survival signals also activate Akt/PKB kinase which phosphorylates (C terminus) and activates Mdm2; the resulting active Mdm2 then triggers the Ubiquitlyation and proteasomal degradation of p53.
Define Mdm2 and Hdm2.
Mdm2 = mouse double minutes 2 HDM2 = Human homolog
How does HDM2 fxn as oncogene
- oncogene (i.e. gain of function leads to transformation)
- Mechanism of transformation involves increased degradation of p53 i.e. loss of a tumor suppressor
What is the Mdm2/HDM2 gene product?
= E3 ubiquitin ligase that targets proteins for proteasomal degradation; attaches ubiquitin to lysine residues of target (i.e. p53)
Where can Phosphorylation of Mdm2 occur
1) N terminus (region
that interacts with p53
2) central acidic region (docking site for binding partners);
multiple P sites in each region
