P53 and cancer

Question 1

What virus transforms cells and induces DNA replication- and what is the protein responsible?

Answer 1

SV40 virus

Protein= Large T

Question 2

What did they see in mammary epithelial cells which had been transformed with SV40>

Answer 2

Saw the SV40 protein in the nucleolus

can stain sv40 antigens using peroxidase staining

Question 3

What experiment involved infecting cells with SV40 and radioactively labelling proteins?

Answer 3

incubate cells in a medium containing radioactive methionine
infected cells in culture with SV40
immunoprecipitate Large T using antibodies
gel electrophoresis and autoradiography to separate the proteins
could see one protein which was bound to Large T
turned out to be P53

Question 4

What experiment determined if P53 was a tumour supressor gene or oncogene? What was wrong with it? What was the marker of cell transformation?

Answer 4

looked at the gene in combination with an oncogene
in one culture- oncogenic ras and a P53 deletion mutant- saw little cell transformation
marker of transformation= anchorage independent growth
in another culture- oncogenic ras and p53 cloned from a tissue culture- saw foci of cell transformation
what was wrong= the cloned P53 had a mutation at the valine at position 135- wasnt wild type P53
When they used WT P53 with the ras= no cell transformation
P53 behaves as a tumour suppressor gene

Question 5

How prevalent are P53 mutations in cancer?

Answer 5

Very common

mutated in at least 50% of cancers

Question 6

Why is P53 different to most tumour suppressor genes?

Answer 6

Knocking it out in mice doesnt result in embryonic lethality

Question 7

Why does knocking T.S genes not usually work?

Answer 7

The genes are involved in negatively regulating cell numbers, and are thus involved in development
knocking them out= embryonic lethality

Question 8

What was the survival like for P53 null mice?

Answer 8

Very poor- most dead before 1 year

heterozygotes= over 70% survival at 1 year +

Question 9

Why did P53 not sit well with knudsens 2 hit theory?

Answer 9

The original experiment involved 3 copies of the P53 gene
2 endogenous copes
1 ectopic copy introduced via a plasmid
doesnt make sense- there was cell transformation when one ectopic P53 gene was mutated

Question 10

How were monoclonal antibodies produced?

Answer 10

Take the spleen out of an animal producing antibodies in response to a protein
fuse the spleen cells with a cancer cell line
=immortal antibody producing cells

Question 11

How did they visualise the half-life of P53?

Answer 11

incubated fibroblasts with radioactive methionine for an hour
they the harvested cells in batches
immunoprecipitated P53 with the monoclonal antibody Pab421
Saw that P53 was rapidly degraded in the cell

Question 12

What happened to the antibodies interaction with P53 when the cells were infected with SV40?

Answer 12

Could not visualise P53 with the monoclonal antibody Pab246
Pab246 could only visualise P53 in a conformation dependent manner- couldn’t detect it when P53 was interacting with Large T

Question 13

What was the evidence that Pab246 was a conformation dependent antibody?

Answer 13

When ELISA was used/ blotting with other antibodies- could see the presence of P53

Question 14

In summary- describe 3 key things learnt about p53 through experiments

Answer 14

1. In cancer the vast majority of P53 mutations were missense
2. P53 had transcriptional activity
3. P53 was a tetramer

Question 15

When it was made clear that P53 was a tetramer- what was understood?

Answer 15

P53 mutations were dominant- explained why in the earlier experiment there was foci with the mutant p53 - even though the cells had 2 endogenous copies of P53
P53 mutations are dominant negative
one mutant unit of P53 leads to the dysfunction of the whole tetramer

Question 16

Why is it better for cancer to have a missense p53 mutation than a null one?

Answer 16

Missense mutation= knock out potentially 15/16ths of P53 function
Null mutation= knock out 50% of P53s function

Question 17

What was P53 thought to be important in?

Answer 17

Maintaining chromosomal integrity

Question 18

How can you distinguish between mutant and WT P53?

Answer 18

By using a panel of conformation-dependent monoclonal antibodies
Pab421= recognises both mutant and WT P53
Pab246= doesnt recognise p53 mutant

Question 19

What happens when you expose skin to increasing time periods of UV light ?

Answer 19

The level of P53 increases

Question 20

What is blocked when stressors are introduced?

Answer 20

The degradation of P53- has a stabilisation effect

Question 21

4 examples of stressors

Answer 21

Lack of nucleotides
Ionizing radiation
Oncogene signalling
Hypoxia

Question 22

Which experiment identified the target genes activated by P53?

Answer 22

Thought that P53 was a transcription factor which activated tumour supressor genes
A p53 null cell line was used- no change of a P53 mutation-
one group kept as null, the other group had WT P53 introduced- its expression was inducible by dexamethasone
Whatever RNA created in the second group which was MISSING in the first group= the tumour suppressor Rna

Question 23

What is subtractive hybridisation?

Answer 23

Take all the RNA from the groups, convert it to DNA
make the DNA single-stranded and allow hybridisation across the groups
when they hybridise they degrade
leaving behind DNA which was expressed in one group but not the other

Question 24

What DNA was found to be activated by P53?

Answer 24

WAF1

Question 25

What did WAF1 turn out to be? Why did it make sense?

Answer 25

P21- a CDKI
CDKIs inhibit CDKs and thus stop the progression through the cell cycle
Made sense- stressors upregulate P21 to arrest the cell cycle and protect the DNA

Question 26

Which other genes were found to be activated by P53?

Answer 26

Genes that:
block angiogenesis 
initiate DNA repair 
are pro-apoptotic 
induce cell cycle arrest

Question 27

What are double minute chromosomes?

Answer 27

2 small chromosomes where one chromosone shouldve been
seen in a subset of mouse sarcomas
they are responsible for the sarcoma phenotype

Question 28

What is the relationship between Mdm2 and P53?

Answer 28

Mdm2 is a ubiquitin ligase and binds directly to and targets P53 for cytoplasmic proteasome degradation
Mdm2 the gene is a target for P53 binding- transcription of mdm2 is regulated by P53

Question 29

What keeps P53 levels low?

Answer 29

Mdm2

Question 30

What keeps P53 levels up?

Answer 30

P14arf gene
exerts a level of negative control on Mdm2- prevents it from binding to p53
keeps P53 in the nucleolus

Question 31

What does genotoxic stress activate?

Answer 31

The protein kinases ATM/ATR

Question 32

What does ATM/ATR do?

Answer 32

They phosphorylate p53 and thus prevent its ubiquitination
ATM= is turned on in response to a double stranded DNA break
ATR= is turned on when DNA replication is arrested

Question 33

What is ataxia?

Answer 33

A genetic disease
homozygous recessive
makes kids very prone to cancer- because their ATM/ATRs are mutated

Question 34

What was ATM/ATR thought to be at one point?

Answer 34

PIP3 kinase- a lipid kinase

due to sequence homology

Question 35

What does P53 inactivation allow cancer cells to do?

Answer 35

Activate oncogenes without apoptosis
Be more tolerant to anoxia- which is common in the core of rapidly growing tumours
Lose chromosomal integrity- as DNA repair pathways arent induced

Question 36

What is a new approach in cancer treatment and why is it possible?

Answer 36

P53 reactivation

The majority of P53 mutations are missense- if we can sort out the structural kink- can reactivate endogenous P53

Question 37

What is PRIMA1? What happens when cells are treated with it?

Answer 37

PRIMA1 is a small molecule that can interact with P53 and change its shape
When a cell line is treated with PRIMA1 the cell cycle profile changes:
-cells start to accumulate more frequently in G2
-host DNA damage is repaired
-large populations form below the G1 population (G0)- apoptotic population of cells