P53 and cancer Flashcards
What is the effect of SV40 on cells
Cells are transformed by it, SV40 proteins are very immunogenic and reside in the nucleus
What is Large T?
A protein derived from SV40, the relevant protein required for transformation and DNA replicaton.
How was P53 discovered
Large T antibodies were added to fibroblasts labelled with 35S methionine
Large T is then immunoprecipitated and see if anything associates with it.
Precipitate was found at 53KD and was named P53
What experiment was done on p53 to see its effect on cellular transformation. What was the problem with this procedure
cDNA encoding oncogenic Ras was co-transfected with either p53 deleted or p53 cloned from tissue cultures.
The number of foci increased in the presence of foci.
The tissue cultured p53 turned out to have a val 135 mutation, WT p53 actually prevented foci formation.
What is p53
A tumour suppressor gene
Why is p53 not a typical tumour suppressor.
Normally tumour suppressor gene deletion result in embryonic lethality in animal models. This is not the case in p53
Normally TSGs have to be completely knocked out to see an effect. Not the case for p53.
Which two antibodies made in David lane’s lab were investigated
pab 421 and pab 246
What immunoprecipitate exp was done in David Lane’s lab and what was the result
Fibroblasts were labelled with pab 241, p53 was found in the nucleus. They were then labelled with 35S methionine for one hour only. Cells were then harvested at 5, 10, 15, 45 and 90 mins. The cells were immunoprecipitated as a function of time with pab421.
By 90 minutes the protein made in the first hour was gone, p53 has a short half life of around 20 minutes.
What was the effect of transforming fibroblasts with SV40 large T on pab 246
pab could no longer detect p53 - due to a change in the epitope?
What three additional interesting things can be said about p53
- Its mutation spectrum ranges almost all cancers, with most mutations being missense
- Introduction of p53 into yeast indicated it as a DNA binding transcriptional activator
- Sucrose density centrifugation showed 53KD, but p53 behaves as if it was four times bigger, i.e. p53 is a tetramer.
What is the effect of one mutation in a p53 tetremer
One mutation has a dominant negative effect. 1/16th of tetramers are likely to be functional in this case.
What was Lane’s theory of the pab 421 and 246 monoclonal antibodies
pab 421 can detect WT p53, pab 246 detects the mutant.
He hypothesised that is there are conversions between WT and mutant, that missense mutations cause a shape change in the epitope that is only recognised by pab 246. hese are therefore conformation specific recognising antibodies.
What 6 factors increase the amount of p53
Lack of nucleotides, UV radiation, ionizing radiation, oncogene signalling, hypoxia, blockage of transcription
What was the inducible promoter experiment to discover p53 target genes
A p53 null cell line was used, a synthetic p53 gene which generates WT p53 in the cells line. It is arranged to operate under an inducible promoter which is bound to a repressor (the repressor is responsive to steroids).
Addition of dexamethazone removes the repressor, this induces p53 expression and their target genes, compared to none in the p53 null cells.
The mRNA is then isolated from both cell lines - reverse transcription then sequence the DNA and compare.
What is subtractive hybridisation
This hybridises mRNA from both cell lines together (after they’ve been transformed into cDNA)
Hybridise one with the other, some won’t hybridise, these are sequenced.
