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BMS 379 Cancer biology > Lecture 8 > Flashcards

Lecture 8 Flashcards

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1
Q

Cancer is a disease of what ?

A

unregulated proliferation

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2
Q

How do they detect proliferating cells?

A

look for presence of cells in s phase

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3
Q

What can be used to induce cells to proliferate ?

A

SV40 virus

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4
Q

What was the model system used to identify the molecular basis for the decision to undertake DNA replication ?

A

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
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5
Q

Why is it easy to create agents against SV40 virus ?

A

SV40 proteins are very immunogenic and reside in the nucleus

- easy to detect

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6
Q

SV40

A

Transforming Virus

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7
Q

David Lane - Discovery of P53

A

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
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8
Q

Relevant viral protein require fro transformation and Inducing DNA replication is ?

A

Large T

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9
Q

What amino acid is present in every protein ?

A

Methionine (Start)

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10
Q

Moshe Oren- Is at tumour suppressor gene or oncogene?

A
  • 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
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11
Q

P53 in cancer

A

Mutated in at least 50 % maybe even 75% of cancers

- the most commonly mutated gene in cancer

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12
Q

P53 - not your typical tumour suppressor gene

A
  • 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
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13
Q

Knudsen conundrum in P53

A

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 **

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14
Q

David lane continuation of previous study

A

MA - map where each MA bound on the POI

- Fine mapped where all the MA bound on P53 found by making truncated p53 mutants

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15
Q

David lane continuation of previous study 2

A

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
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16
Q

P53 half life

A

20 mins

17
Q

Pab 246 - experiment

A

Lane group looked at immunostaining when you have or havent transformed fibroblast with SV40 Large T
** cant really see P53 **

18
Q

Pab246 findings

A

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 ***

19
Q

Pab 246

A

Conformation specific antibody

20
Q

P53 mutation spectrum

A
  • 75 % missense
21
Q

P53 DNA binding/Transcriptional activation

A

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

22
Q

What kind of molecule is P53 ?

A

Tetramer

- sucrose density centrifugation

23
Q

Why is p53 tetrameric status important?

A

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

24
Q

p53 mutants

A

Dominant Negative

25
Q

p53

A

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
26
Q

Experiment - David Lane and Peter Hall

A

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

27
Q

What stabilises P53 levels ?

A
  • Lack of nucleotides
  • Uv radiation
  • Ionising Radiation
  • oncogene signalling
  • Hypoxia
  • blockage of transcription

BMS 379 Cancer biology (18 decks)

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