Lecture 8 p53 and Cancer

Question 1

What is the effect of infecting cells with polyomavirus SV40

Answer 1

This causes transformation of the cells and an induction of proliferation and DNA replication

Question 2

Which immunogenic protein isolated from SV40 viruses mediate their transforming ability

Answer 2

Polyoma large T

Question 3

How was p53 identified

Answer 3

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

Question 4

Describe the experiments of Moshe Oren that lead to the idea that p53 was an oncogene

Answer 4

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.

Question 5

What flaw in Oren’s experiments with p53 lead to the wrong assumption that p53 was an oncogene

Answer 5

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

Question 6

What kind of gene is p53 and what was the evidence for this

Answer 6

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

Question 7

What is the incidence of p53 mutations in cancer

Answer 7

p53 is mutated in 70-80% of almost all cancers indicating it is a significant tumour suppressor gene that is frequently mutated

Question 8

What is the effect of tumour suppressor gene knockout

Answer 8

Knockout of tumour suppressor genes is embryo lethal

Question 9

What is different about the p53 knockout mouse

Answer 9

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

Question 10

What is meant by the Knudson conundrum caused by p53

Answer 10

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

Question 11

How was it shown where p53 was localised to

Answer 11

P53 was found to be localised to the nucleus using monoclonal antibodies against the protein

Question 12

Explain the pulse chase experiments used to determine the half-life of the p53 protein

Answer 12

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

Question 13

What is the half-life of p53

Answer 13

20mins

Question 14

Which p53 monoclonal antibody was used in the pulse chase experiments which region of the protein does it recognise

Answer 14

PAb421 – this recognises the C terminus

Question 15

What do the results below infer about p53 expression

Answer 15

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

Question 16

Which monoclonal antibody could no longer detect p53 in IHC experiments after the cells were infected with SV40

Answer 16

PAb246

Question 17

What did quantitative ELISA and Western blot experiments carried out using PAb421 show about p53 levels before and after infection with SV40

Answer 17

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

Question 18

What hypothesis was there for the fact that PAb421 couldn’t detect p53 in cells infected with SV40

Answer 18

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

Question 19

What is significant about the mutation spectrum of p53

Answer 19

75% are missense mutations with very few deletions insertions frameshifts and non-sense mutations seen

Question 20

How was it shown that p53 is a transcription factor capable of self-repression

Answer 20

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

Question 21

How was it shown that p53 is a tetramer

Answer 21

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

Question 22

How was the Knudson conundrum of p53 solved

Answer 22

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

Question 23

P53 maintains genome integrity this is lost in cancer T or F

Answer 23

T

Question 24

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

Answer 24

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

Question 25

Which form of p53 was being recognised by PAb421

Answer 25

Wild type p53

Question 26

Which form of p53 wasn’t being recognised by PAb246

Answer 26

Mutant p53

Question 27

What is the overall effect of SV40 infection on p53

Answer 27

SV40 binds p53 and changes its shape so it adopts a mutant conformation.

Question 28

What is the relationship between missense mutations and SV40 infection with regards to p53

Answer 28

The conformation of p53 induced by transforming proteins during an SV40 infection is the same as the conformation adopted as a result of missense mutations in the p53 protein itself

Question 29

What is the effect of DNA damaging agents on p53

Answer 29

In response to things that induce DNA damage you stabilise p53 increasing its half-life and it effects in triggering transcription

Question 30

What sort of factors increase p53 stabilisation

Answer 30

Lack of nucleotides UV and ionizing radiation oncogene signalling hypoxia and a blockage of transcription

Question 31

What is the role of p53

Answer 31

It’s a transcription factor

Question 32

Describe how the first p53 target genes were identified

Answer 32

A synthetic p53 construct was introduced into a p53 null cell line. This synthetic p53 gene was downstream of an inducible promoter that binds a steroid-sensitive repressor. In the absence of steroid hormones p53 expression is repressed and therefore so too are its target genes. However upon the addition of dexamethasone the repressor is displaced and there is subsequent transcription of p53 which can then in turn transcribe its target genes. Then the mRNA transcriptome from two populations of cells were extracted one population treated with dexamethasone and the other that hadn’t. This would allow for comparison of the genes upregulated by an activation of p53. To do this subtractive hybridisation was carried out. Firstly cDNAs were synthesised from all of the extracted mRNA. Then the two populations of cDNA were hybridised so that complimentary cDNA from the dexamethasone treated cells would hybridise with cDNA from the untreated cells. This would happen in the genes that are expressed in both conditions. Then all of the dsDNA was digested with an enzyme that specifically recognises dsDNA. From this it was then possible to isolate out the remaining ssDNA which corresponds to the genes upregulated with dexamethasone treatment.

Question 33

WAF1 was one of the first p53 genes identified what was significant about its expression in the cell line with the inducible promoter

Answer 33

Its expression in the cell line was in a dexamethasone-concentration-dependent manner

Question 34

What was the significance of the WAF1 gene

Answer 34

WAF1 turned out to be the Cdk inhibitor (CKI) p21Cip1. This made perfect sense as the CKIs are important in inhibiting Cdks and preventing cell cycle progression

Question 35

How does induction of p53 lead to cell cycle arrest

Answer 35

Induction of p53 results in cell cycle arrest through the inhibition of cdks by WAF1

Question 36

What are the 4 processes affected by p53 stabilisation

Answer 36

Cell cycle arrest induction of DNA repair genes blockage of angiogenesis and a promotion of apoptosis

Question 37

Give some examples of p53 target genes

Answer 37

Growth arrest genes – p21Cip1 and 14-3-3 DNA repair genes – p53 inducible ribonucleotide reductase and XPC regulators of apoptosis – Bcl-2 and NFκB anti-angiogenic proteins – thrombospondin

Question 38

What happens under normal conditions where p53 isnt stabilised

Answer 38

The p53 half-life is kept extremely short by a futile life cycle. Here it is made and then rapidly ubiquitinated by the E3 ubiquitin ligase mdm2 this marks it for degradation by the proteasome.

Question 39

How is a stabilisation of p53 achieved during cell cycle stressors

Answer 39

In response to cell cycle stressors such as DNA damage lack of nucleotides and hypoxia the ATM/ATR kinases are turned on and phosphorylate p53. Phosphorylation of p53 stabilises it and prevents its ubiquitination and degradation allowing it to cause its downstream effects

Question 40

How would p53 inactivation prove advantageous for incipient cancer cells

Answer 40

Inactive forms of p53 enable oncogene activation without apoptosis a higher tolerance to anoxia and a sloppy oversight of chromosome integrity (encouraging cancer cell evolution)

Question 41

What is PRIMA-1 and how does act as a potential cancer treatment

Answer 41

PRIMA-1 covalently binds to the mutant core domain of p53 and shifts it back to its wild type conformation. It hence acts as a gain of function drug that allows p53 to function

Question 42

Draw a diagram of a typical cell cycle FACS trace

Answer 42

See completed diagram below

Question 43

Below is a FACS trace from a population of cells describe what this trace shows and explain what this implies

Answer 43

This trace shows a population of cells undergoing apoptosis this is indicated by the sub-G1 DNA content of some of the cells seen in the left of the graph. This sub-G1 DNA content is due to the activation of nucleases which have begun to breakdown DNA hence resulting in a relative DNA content below 1