Lecture 8 p53 and Cancer Flashcards
What is the effect of infecting cells with polyomavirus SV40
This causes transformation of the cells and an induction of proliferation and DNA replication
Which immunogenic protein isolated from SV40 viruses mediate their transforming ability
Polyoma large T
How was p53 identified
Antibodies were raised against large T. Fibroblasts were then radioactively labelled with 35S methionine before an immunoprecipitation experiment was carried out using beads coated with the anti-large T antibodies. This would elute large T from cells infected with SV40 together with any potential binding partners of the protein. The proteins eluted from the IP experiment were then ran on a gel and exposed to a radioactive sensitive film. A band for the large T protein was seen at 94kDa as well as another band at around 53kD. These bands were only present in the cells that had been infected with SV40 and not control cells. Isolation of the protein from the band at 53kDa and subsequent cloning lead to the identification of p53
Describe the experiments of Moshe Oren that lead to the idea that p53 was an oncogene
Fibroblast cells were transfected with an oncogenic ras to elicit transformation and a degree of anchorage-independent growth. The cells that were used also had a p53 deletion so in one population they introduced p53 cloned from tissue culture cells. This increased the number of foci within the cell population and lead to the idea that p53 might be an oncogene due to the synergistic effect with ras.
What flaw in Oren’s experiments with p53 lead to the wrong assumption that p53 was an oncogene
The form of Ras that he transfected into fibroblast along with oncogenic Ras was in itself a mutant version of the p53 gene. This possessed the valine 135 mutation that renders the protein non-functional
What kind of gene is p53 and what was the evidence for this
P53 is a tumour suppressor gene. Evidence of this came from transfected fibroblasts with oncogenic Ras and then wild type p53. This lead to a suppression of foci formation that was seen in cells transfected with oncogenic Ras alone
What is the incidence of p53 mutations in cancer
p53 is mutated in 70-80% of almost all cancers indicating it is a significant tumour suppressor gene that is frequently mutated
What is the effect of tumour suppressor gene knockout
Knockout of tumour suppressor genes is embryo lethal
What is different about the p53 knockout mouse
Unusually for a tumour suppressor gene knockout of p53 is not embryo lethal. These mice do survive although have a drastically reduced life span due to increased incidence of sarcomas and lymphomas
What is meant by the Knudson conundrum caused by p53
Normally tumour suppressor genes have to be completely knocked out (both copies) to see an effect. However introduction of 1 non-functional p53 causes the effects expected when both copies of a tumour suppressor gene are knocked out. even when 2 functional copies remain. For example fibroblasts transformed with Ras and val135 p53 have 3 copies of the p53 gene 2 wild type and one val135 mutant. However these cells still see foci anchorage-independent growth and other features indicative of transformation
How was it shown where p53 was localised to
P53 was found to be localised to the nucleus using monoclonal antibodies against the protein
Explain the pulse chase experiments used to determine the half-life of the p53 protein
Labelled fibroblasts with 35S methionine and incubated the cells for one hour only. After this point radioactive methionine was removed and replaced with non-radioactive methionine. Cells were then incubated again and harvested at a number of different time points. They then immunoprecipitated p53 as a function of time using the PAb421 anti-p53 antibody. Over time the immunoprecipitated p53 becomes more and more faint until vanishing after 90mins. This indicated the p53 was extremely unstable with a very short half-life
What is the half-life of p53
20mins
Which p53 monoclonal antibody was used in the pulse chase experiments which region of the protein does it recognise
PAb421 – this recognises the C terminus
What do the results below infer about p53 expression
This data shows that p53 couldn’t be detected once infected with SV40 using the PAb 246 monoclonal. This implies that p53 disappears once cells are infected with SV40
Which monoclonal antibody could no longer detect p53 in IHC experiments after the cells were infected with SV40
PAb246
What did quantitative ELISA and Western blot experiments carried out using PAb421 show about p53 levels before and after infection with SV40
The total amount of p53 was exactly the same in cells that had been infected with the virus and those that had not. Hence p53 is seemingly present at the same total levels in cells regardless of infection with SV40
What hypothesis was there for the fact that PAb421 couldn’t detect p53 in cells infected with SV40
Although once infected with SV40 the PAb246 could no longer bind to p53 other antibodies could. Hence this means that the epitope which PAb246 was recognising must be no longer present/exposed in the infected cells
What is significant about the mutation spectrum of p53
75% are missense mutations with very few deletions insertions frameshifts and non-sense mutations seen
How was it shown that p53 is a transcription factor capable of self-repression
The GAL4 expression system was used whereby the GAL4 promoter was positioned upstream of an acetyl transferase. The addition of p53 increases expression of the GAL4-driven gene. Interestingly the first 70 amino acids were found to be critical for its transcription factor activity. Truncation mutations in p53 increased it transcriptional activity. This indicated that wild type full length p53 is seemingly repressing itself to regulate its activity
How was it shown that p53 is a tetramer
It was known from IP and cloning experiments that the polypeptide for p53 was 53kDa. However sucrose-density centrifugation used to separate the protein out showed the p53 came out as a 200kDa protein. This implied that p53 is a tetramer
How was the Knudson conundrum of p53 solved
As p53 was found to be a tetramer it was thought that mutant p53 may be having a dominant negative effect. It was found that for p53 to be functional all 4 subunits need to be wild type. If a p53 mutation subunit is incorporated into the tetramer the whole tetramer will be non-functional. Hence if you introduce a non-functional copy of the p53 protein it will disrupt the wild type p53 copies. This is why p53 didn’t behave like a tumour suppressor gene in vitro
P53 maintains genome integrity this is lost in cancer T or F
T
What is the explanation for why some of the monoclonal antibodies for p53 weren’t able to recognise it once cells had be infected with SV40
The anti-p53 monoclonal antibodies were conformation-specific antibodies. These PAbs can only recognise the p53 protein in certain conformations. This implied that once infected with SV40 the conformation of p53 is altered
