Lecture 8 Flashcards
Cancer is a disease of what ?
unregulated proliferation
How do they detect proliferating cells?
look for presence of cells in s phase
What can be used to induce cells to proliferate ?
SV40 virus
What was the model system used to identify the molecular basis for the decision to undertake DNA replication ?
Polyomavirus SV40
- viral infection promotes DNA replication in host
- idea to understand the mechanism viruses use might tell us how mammalian cells control DNA replication
Why is it easy to create agents against SV40 virus ?
SV40 proteins are very immunogenic and reside in the nucleus
- easy to detect
SV40
Transforming Virus
David Lane - Discovery of P53
infect with SV40 and immunoprecipitate with Large T
- made Antibodies to Larget T
- incubate all cells with radioactive methionine
- immunoprecipitated Large T
- electrophoresis and exposure to film
- film turns black where banding
- fist identification of a cellular protein that specifically interacts with SV40 protein Large T
Relevant viral protein require fro transformation and Inducing DNA replication is ?
Large T
What amino acid is present in every protein ?
Methionine (Start)
Moshe Oren- Is at tumour suppressor gene or oncogene?
- cloned P53 from tissue culture cells
- Agar gel
1. Oncogenic ras + P53 deletion mutant
2. Oncogenic Ras plus P53 cloned from tissue culture cells - increase in number of foci formed - synergy with Ras?1 - P53 contains Val135 mutation
3. Oncogenic Ras plus P53 WT - clearly behaves as a TS gene - suppressed transformation phenotype seen and produced by oncogenic RAs
P53 in cancer
Mutated in at least 50 % maybe even 75% of cancers
- the most commonly mutated gene in cancer
P53 - not your typical tumour suppressor gene
- experimental deletion of most TS results in embryonic lethal because many TS are negative regulators of cell numbers
- double KO > no progeny (normally)
- P53 -/- or p53 -/+ survival drops dead before year
but survival
Knudsen conundrum in P53
Notion 1 or 2 hits sufficient to get emergence of cancer in TS
- ohrens experiment
primary fibroblast used have 3 sources of P53
2 endogenous and 1 ectopic mutant
** Doesnt obey Knudsens rule for TS **
David lane continuation of previous study
MA - map where each MA bound on the POI
- Fine mapped where all the MA bound on P53 found by making truncated p53 mutants
David lane continuation of previous study 2
Labelled fibroblasts with methionine for 1 hour only - so on the autoradiogram you only see cells made in last hour
- harvest cells at indicated time
- immunoprecipitate P53 with Pab421
- band disappears tells you about turn over not levels of P53 protein in cell - half life of 20 mins
P53 half life
20 mins
Pab 246 - experiment
Lane group looked at immunostaining when you have or havent transformed fibroblast with SV40 Large T
** cant really see P53 **
Pab246 findings
Detect P53 in a manor that is Selective for whether or not Large T antigen was present
** lead on to the suggestion that there is a subset of MA that are conformation specific - recognise P53 in normal conformation but doesnt if it adopts different ***
Pab 246
Conformation specific antibody
P53 mutation spectrum
- 75 % missense
P53 DNA binding/Transcriptional activation
Transcriptional reporter system -
galactose in yeast looking at out put of target gene when you make P53 in yeast
- make lots of transcriptional promoter and ask what impact they have on P53 down stream of promoter
- Yeast 1 hybrid
What kind of molecule is P53 ?
Tetramer
- sucrose density centrifugation
Why is p53 tetrameric status important?
goes back to Ohren experiment p53 mutant dominance
- anyone of the monomers that form the tetramer broken all four broken - dominant effects - heterozygote doesn’t work because you need all 4 subunits to be functional
p53 mutants
Dominant Negative
p53
Guardian of the genome
- looks after integrity of the chromosome ( lost in cancer)
- use antibodies that are confirmation specific to distinguish between Wt and mutant
Experiment - David Lane and Peter Hall
Expose skin to UV light
- blot it with actin/ p53
over time and exposure there is an increase in p53 - becomes stabilised in response to ultraviolet light
What stabilises P53 levels ?
- Lack of nucleotides
- Uv radiation
- Ionising Radiation
- oncogene signalling
- Hypoxia
- blockage of transcription
