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Canc Biology > P53 lecture > Flashcards

P53 lecture Flashcards

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1
Q

How was P53 discovered?

A
  • used SV40 virus - cells transformed when injected with SV40
  • SV40 viral antigen is called polyoma large T
  • antibodies were made for polyoma large T and RA methionine was added to fibroblasts - P53 immunoprecipitated with largeT
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2
Q

does P53 have synergy with Ras?

A

No

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3
Q

what were the steps that showed that P53 does not have synergy with Ras?

A
  • when oncogenic Ras was with a deletion mutant of P53 some transformation occurred
  • When oncogenic ras was with P53 cloned from tissue culture cells there was even more foci/transformation - does ras have synergy with P53?
  • was later found that this form of P53 in the cells was a mutant form of P53 (Val135)
  • When oncogenic Ras was with WT P53 there was no transformation of cells
  • this showed that P53 is a tumour suppressor gene and does not have synergy with ras
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4
Q

what type of gene is P53?

A

tumour suppressor gene

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5
Q

What staining can be used to visualise SV40 antigens?

A

peroxidase

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6
Q

what gene codes for the P53 protein?

A

TP53

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7
Q

How often is P53 mutated in human cancers?

A

P53 is frequently mutated in human cancers - almost all cancer

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8
Q

Why is P53 not a typical tumours suppressor gene?

A
  • usually when TS genes are null it results in embryonic lethality ( as TS genes control/supress cell numbers)
  • But when there is a P53 null mouse it develops into an adult mouse
  • doesn’t obey knudsons 2 hit hypothesis
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9
Q

Why does P53 not follow knudsons 2 hit hypothesis rule for tumour suppressor genes?

A

-primary mouse fibroblasts which were examined have 3 sources of P53 (1 exongenous from virus, and 2 endogenous)
-yet when a mutant form of P53 is introduced (from virus) there is an effect despite there being 2 WT copies still
(usually all genes must be defective for a TS gene to have an effect)
-even heterozygous P53 has an effect

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10
Q

what happens to mice that are null for P53 on kaplan meier plot?

A

mice die within a year

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11
Q

what is the disease caused when mutant TP53 is carried in the germline?

A

Li-fraumeni syndrome

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12
Q

What is Li-Fraumeni syndrome?

A

A rare disorder which increases the risk of developing cancers especially in children and young adults - caused by mutated TP53 i the germline.

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13
Q

what experiment looked at the mystery of the disappearing protein?

A

fibroblasts were labelled with radioactive methionine for 1 hour

  • cells were harvested at set time points
  • immunoprecipitate P53 with pab421
  • pulse chase experiment - add in non RA methionine - replaces RA methionine with normal methioine
  • can work out how long protein survives for
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14
Q

what is the half like of P53 and what does this say about it?

A

half - life is 20/30 minutes

- P53 goes away over time, shows this is an unstable protein as it turns over very quickly

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15
Q

how many kDa is P53?

A

53 kDa

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16
Q

what experiment showed the mystery of the reappearing protein?

A
  • infect cells with SV40 large T virus
  • AB pab246 cannot recognise P53 in transformed cells
  • ELISA and blot with pab246 and other monoclonal ABs- found there was exactly the same amount of P53 is normal and transofmred cells
  • P53 has a different structure/ is a different epitope in transformed cells (this epitope cannot be detcetd by pab 246 but can be detected by other ABs, e.g. pab 421
17
Q

what is the most frequent mutation type in P53?

A 
missense mutation 
(single base change/ point mutation which changes the amino acid)
18
Q

what kind of facotr can P53 act as and how was this shown?

A
  • transcription facotr
  • shown in yeast using the GAL4 promoter system
  • fuse piece of P53 with GAL4
  • more gene product when P53 fused with GAL4 - gene transcription increases by 20 fold
19
Q

can P53 repress itslef?

A

YES

  • P53 is a TF which is capable of repressing itself
  • when P53 is truncated, its expression increases
20
Q

what kind of structure is P53? (e.g. monomer, dimer?)

A

P53 is a tetramer

21
Q

how was it sown that P53 is a tetramer?

A

It was shown that P53 is a tetramer by sucrose density centrifugation

  • protein moves through sucrose and the way it moves is due to MW
  • (has marker proteins that know the MW of)
  • P53 is not behaving as a monomer it behaves as a ~200kDa protein - is behaving as a tetramer
22
Q

why is the tetrameric stautus of P53 important?

A

as it has a dominant negative effecr

  • all 4 WT subunits atre required for P53 to be functional
  • 16 different combinations but only 1 is all 4 WT
  • explains why when mutant P53 is intoduced it has a large effect
23
Q

why are ABs to P53 (pabs) unusual?

A

they are conformation specific

  • e.g. pab246 only binds WT conformation
  • pab421 binds any isoform/ conformation
24
Q

what did an experiment done by Hall et al in 1993 show?

A

P53 becomes stabilised in human skin when exposed to high levels of UV
- transcription increases when P53 levels are stabilised

25
Q

what is the name of the DNA binding sequence on P53?

A

polygrip

26
Q

what can cause an increase in P53 in cells?

A
  • lack of nucleotides
  • UV radiation
  • ioising radiation
  • oncogene signalling
  • hypoxia
  • block of trnascription
  • cell damage/stress
27
Q

what experiment was used in 1990 by El diery et al to look for P53 target genes?

A
  • P53 null mice used
  • introduced synthetic P53 gene which could be induced when repressor bound to promoter site is removed (responsive to steroids)
  • when dexamethazone (steroid) is injected, repressor comes off of promoter site - causes expression of of P53
  • look for genes transcribed - isolate and compare mRNA
28
Q

what technique was used to look for/sequence the target genes of P53 in mouse exp?

A

subtractive hybridization exp

  • eliminate genes the 2 cell lines (normal and dexamethazone conditions)
  • separate out differences and sequence RNA into DNA
29
Q

what was the first P53 target/downstream gene identified in mouse exp?

A

WAF1

expressed in a dexamethazone conc dependent manner

30
Q

what did WAF1 turn out to be when it was sequenced?

A

P21
- a small cyclin dependent kinase inhibitor (CKI)
(can arrest the cell cycle)

31
Q

What are some of the roles of the genes that P53 actgivates?

A
  • cell cycle arrest
  • promote DNA repair
  • block angiogenesis
  • promote apoptosis
32
Q

what is the ubiquitin ligase that causes the proteolysis of P53 in unstressed conditions?

A

NDM2

MDM2

33
Q

what kinases are switched on in response to genotoxic stress?

A

ATM/ATR kinases

  • phosphorylate P53 subunits
  • stabilises P53
  • increases trans of P53 target genes
  • induce apoptosis, DNA repair and cell cycle arrest
34
Q

How does P53 inactivation give an advantage to cancer cells?

A
  • oncogene activation can occur without apoptosis
  • higher tolerance to anoxia
  • sloppy oversight of chromosome integrity/damagwe
  • allows cancer cells to evolve without being targetted
35
Q

When PRIMA-1 is used what does FACs analysis show?

A
  • when PIMA-1 is used there is more cells in the SUB-G1 phase (with less DNA as nucleases have been released that destroy DNA - apoptosis induced by activated P53 induced by PRIMA-1)
36
Q

What does PRIMA-1 do to reactivate P53?

A

covalently binds to mutant core domain and tweaks back into shape

37
Q

list some P53 target genes

A 
P21cip (CKI)
MDM2
PTEN 
APAF1
PERT
BCL2
TSP1 (antiangiogenic)

Canc Biology (15 decks)

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