L10 - Genomic Instability Flashcards by Jack Corston

Describe the disovery of p53

In 1975 - thought to be the basis of the decision to replicate the DNA

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What virus was used to help discover p53

Papovirus - SV40

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Describe the normal distribution of SV40 protein

Reside in the nucleus

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Advantage of SV40 proteins

Immunogenic - they are easy to raise antibodies against

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What is the relevant viral protein from SV40

Large T

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Describe what happens when you transfect cells with viral SV40

What is the experimental method required to see this

Can see that proteins are constantly undergoing protein synthesis

Shown through the addition of a radio-AA

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What was the next direction after the SV40 transfection

To ask wehther large T interacted wth any other host proteins

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Results of immnoprecipitation experiemnts of cells TF with SV40 large T

In presence of antiserum or antiserum from control animal pre-immunisation
Only immunopreciptate in transformed SV40 cells where antibody present

Band around 54kD = p53

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Purpose of a cellular transformation assay

Allows you to determine if cell is transformed in the presence of an oncogene

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How would you tell if a cell had been transformed

If the cell shows anchorage independent growth … transformation … clone into an immune compromised mouse

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Ras + P53 mutant

Normal number of colonies

CONTROL

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Ras+P53

P53 FROM TISSUE CULTURE CELLS

Increased numbr of colonies … looks like p53 acting as an oncogene

BUT

Tissue culture cancer cell line - vast majority of p53 has been lost due to genomic instability

(In the case from the paper there was a Val135 mutation)

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Ras + WT (true) P53

No colonies
Onogenic function of Ras has been supressed

P53 BEHAVES AS A TUMOUR SUPRESSOR GENE

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What cancers display a p53 mut

ALL

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What happens usually when TSGs are deleted in the embryo

What is done to get around this?

Is this the case for p53

Embryonic lethality
- Since many TSGs are regulators of cell numbers

Conditional KO approaches

Not the case for P53

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Why are the Kaplan Meier plots for p53 mutations unusal?

If P53 is a TSG would expect to see Knudsens rules followed… but they aren’t

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What is an epititope map

Pannel of antibodies that recognise p53 but each recognises it at a different site (epitope)

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Describe the experiments that lead t the conclusion of p53 having a short half life

Label cells with radio-methionine
Harvest cells at 1 hour
Blot for p53 with Pab421
Can see after around 45 minutes alsmost all 421 expression is lost

p53 HAS A HIGHTURNOVER RATE

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Descibe the effect of LargeT/p53 interaction has on Pab426 binding

With large T present p53 cant bind anymore

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Describe the experiments looking at how much p53 was in the cells

Control vs large T TF

In normal 421 detects p53

But in SV40 transformed cells - 246 detectable p53 in non transfect but in SV40 246 cant detect p53

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Is it the levels of p53 that changes in the SV40 transformed cells?

No…

Using a-p53 for a different epitiope … shows there is equal ammounts of p53

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What sort of mutations occurs in p53

What is unusual about this?

Most are missense mutations

There is an unexpected bias in the mutations

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Domains within p53 affect transcriptional activation of ….

Gal4

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Describe the experiments looking to see if p53 engaages with DNA as a transcritpion factor

STRUGGLES TO DEMONSTRATE DNA BINDING

If have DNA binding domain and transcriptional activating domain = lots of gene expression

Take VP16 and fuse p53 to gal4 construct

If just use N-terminal p53 can show transcriptional activation

Suggests part had transcriptional activation

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Evidence that p53 acts as a tetramer

Migrates as a 45kd and 145kd protein during glucose sucrose density gradient centrifugation

What does the tetrameric strucuture explain about p53 acting unusally as a TSG

In hets Unlikely to get 4 subunits of total WT (only 1/16 chance) Likely to have AT LEAST one mutant subunit (15/16)

Wt P53 is tthe

Guardian of the genome

mutant p53 at Pab246 site...

Loses its guardian role

When is the guardian role of p53 lost?

Lost in the presence of large T or through missense mutations

What happens to p53 levels if you irradiate the skin withUV How was this seen

Increase in the levels of p53 Western blot experiments

Describe an experiment to see what genes are promoted by p53

Null cell line is easy to come by since p53 is commonly mutated Insert p53 into null line under control of glucocorticoid promoter Apply dexamethasone ... Look for differences in the transcriptional output ISOLATE mRNA Subtractive hybridisation Essentially to identify which genes arent present

What was a gene that p53 promotes

WAF1

WAF1 aka

p21

What is p21 Effects of p21

CDK inhibitor Inhibits CDK4/2/1 Controls the G1 --> S phase progression through p53

Types of genes that p53 upregulates

Growth arres genes DNA repair genes Regulators of apoptosis Anti-angiogenic proteins

Specific alarm signals provoke p53 mediated

Inhibition of proliferation | Triggering of apoptosis

Cytostatic and pro-apoptotic functions of p53 represent a threat to incipient cancer cells for which the following are necessary conditions

Hypoxia Genomic damage Signalling excess

Inactive forms of p53 enable

Oncogene activation wo apoptosis Higher tolerance of anoxia Sloppy oversight of chromosomee inegrity

What is the double minutee phenotype

Double minute chromosomes seen in fibroblasts NIH-3T3 Cells able to spontaenously transform and produce tumours Identified and cloned genes OE in these cells

What genes are OE in the double minute phenotype

Mdm1-3

OE Mdm1-3 sufficient to cause

Transformation

Coprecipitation of ___ and ____

Mdm2 and p53

Mdm2 plays an important role in ...

Inducing the destruction of p53

Increasing mdm2 lead to ...... in a ...

P53 not being present | CONCENTRATION DEPENDENT MANNER

What was the control when looking at the p53 / mdm2 relationship

Using mutant p53 which is lacking the N-terminal

Mdm2 is a

Ubiquitin ligase

What does Mdm2 do to p53

polyubiquitinate marking for proteasomal degradation

if you increase Mdm2 ... p53 ______ What is Mdm2 acting as`

p53 will decrease Mdm2 acting here as an oncogene

Give some examples of dependency points ... explain each one Are dependency events absolute?

Bacteriophage assembly Later component assembly dependent on the correct assembly of earlier ones Drosophila polytene chr Huge - exist since multiple reps without division , SHOWS depdency events are not aboslute Polyploidy Multipel chr per cell SHOWS depdency events are not aboslute NO

Outline the paradigm for S/M checkpoint control in Yeast

Cells lacking in Rad9 healthy in the absence of extrinsic interference

What is CDC9 What is the effect of losing Cdc9?

DNA ligase - responsible for sealing the nick between okazaki fragments WO Cdc9 cant get a sigar phosphate backbone all of the way through the molecule

Describe the mutation in the Yeast paradigm What were the mutants crossed with

Temperature dependent Cdc9 mutation Too high - null Permissive - Cdc9 will work Done this was since conventional Cdc9 KO would be lethal Then cross Cdc9 mutants with Rad9 mutants

Descirbe the Cdc9 ts mut/ Rad9 What would you expect to seen ... what is actually seen At the permissive temperature At the restrictive temperature

Normal Would expect them to be stuck in S phase Since checkpoint contols should prevent the advance of the cell cycle when replication is incomplete BUT ACTUALLY SEE CELLS ADVANICING INTO M PHASE

Describe the viability determination of these cells

Wt Cdc9 ts - stuck in S phase but still viable Rad9 - viable Cdc9ts Rad9 - because no Rad9 cells going into G2 and M without properly replicated DNA

Rad9 is the ....

Founder of the S/M checkpoint in yeast

Describe the experiments to demonstrate the S/M checkpoint in mammalian cells

Hamster cells with ts mut At restricitve temp ith DNA rep inhibitor of colchicine (blocks cells in M) Restrive - fragmentation of chromosomes caused by improper segregation Permissive - condensed chromosomes

Loss of checkpoint control may also be induced with

Caffenine

All metozoan systems have a ______- componenet at the S/M checkpoint

Caffeine sensitive

What does caffeine interfere with

Transducers and effectors

Describe the replication fork sensors

At the replication fork ... Stalling due to lesion leads to the assembly of a complex sensor system Important to ensure integrity during replication Occurs ALOT

ATR is ________ sensnitve and is related to ____

Caffeine sensitive | Rad3

ATR is a

Protein kinase - very complex strucutre and is an atypical protein kinase TRANSDUCTION DEVICE WHICH IS CAFFEINE SENSITIVE

Describe the mechanism involving ATr that occurs following DNA damage

Replication stress leads to ATR being turned ON Assembly of the sensor Turns on ATR Turns on Chk1 +wee1 , -cdc25 Cdk1 is OFF and entry into mitosis is blocked

What does the inherited loss of checkpoint proteins demonstrate

The role of checkpoints in supressing cancers (spontaneous and inherited)

Checkpoint signalling is essential for

P53 function

What is cisplatin

Chemotoxic agent | Is a chemotherapy drugs used to solid tumours - induces apoptosis

Describe what happens when cisplatin is applied

p53 is phosphorylated at serine-15 - N terminal P53 (criticial for Mdm2 interaction) - this prevents the binding of Mdm2 and p53 is not degraded

Describe what happens when a dominant negative form of ATR is used

No phos and activation of p53 | Mdm2 able to ubiquitanted and degrade p53

L10 - Genomic Instability Flashcards

(68 cards)