Cancer Bio (Dan Lecture 4 - p53)

Question 1

p53 mutagenesis is common in cancers. True or false?

Answer 1

True

Question 2

p53 mutation levels are the same in all cancer types. True or false?

Answer 2

False

Question 3

p53 knockout mice showed what 2 main differences?

Answer 3

• Reduced survival

- Enhanced tumourigenesis

Question 4

What is the structure of native p53?

Answer 4

• A tetramer in its native state
• Expressed as a monomer (single gene)
• 4 copies produce native state

Question 5

Name some factors that can induce p53 formation

Answer 5

• lack of nucleotides
• UV radiation
• Ionizing radiation
• Oncogene signalling
• Hypoxia
• Blockage of transcription

Question 6

Name some processes that p53 regulates

Answer 6

• Cell cycle arrest
• DNA repair
• Blockage of angiogenesis
• Apoptosis

Question 7

What is p53’s normal function?

Answer 7

To upregulate expression of target genes e.g p21 gene

Question 8

What is the Mdm2 gene and how does it function to regulate p53 levels

Answer 8

• Gene products acts as negative feedback mechanism:
• p53 upregulates Mdm2 gene
• Mdm2 protein moves back to nucleus and binds p53
• Prevents its activation by preventing phosphorylation
• Complex exported from nucleus to cytoplasm, ubiquitinated and degraded by proteasome

LOOK AT DIAGRAM ON SLIDE TO CONFIRM

Question 9

Is p53 a stable protein? What is its half life?

Answer 9

Very unstable protein

- 20 min half life

Question 10

How can p53 prevent degradation by mdm2 and therefore increase its half life?

Answer 10

• Phosphorylation of p53 at the N terminus in the mdm2 binding domain
• Prevents mdm2 binding
• Increases half life

Question 11

What affect will p53 knockout have on cell viability?

Answer 11

• loss of p53 HUGELY increases cell survival after DNA damage
• Opposite to what was observed in the mice
• No p53 in cells = loss of apoptosis = cell survival
• In mice: No p53 = no apoptosis = damaged DNA allowed to replicate leading to tumour formation therefore no p53 = mice death

Question 12

In addition to p21, what other important protein is is upregulated by p53 as a result of UV induced DNA damage?

Answer 12

p48

Question 13

What is p48?

Answer 13

• A protein subunit of the UV-DNA damage-binding protein (UV-DDB)
• Otherwise known as DDB2

Question 14

What do intrinsic and extrinsic apoptotic pathways mean?

Answer 14

Intrinsic - apoptosis induces by intracellular components

Extrinsic - apoptosis induced by extracellular signals

Question 15

What is the main intrinsic pathway of apoptosis?

Answer 15

BAX signalling:

1) p53 transcriptionally upregulates BAX
2) Bax increases permeability of mitochondrial membrane by opening channels
3) Release of pro-apoptotic cytochrome c from mitochondria
4) Cyt c associates with Apaf-1
5) Procaspase 9 binds to complex forming apoptosome

6) Apoptosome causes cleavage and acitvation of
executioner caspases

7) Executioner caspases cleave death substrates resulting in apoptosis

Question 16

What is the main extrinsic pathway of apoptosis?

Answer 16

Apoptosis can be activated by transmembrane death receptors e.g FAS:

1) p53 transcriptionally upregulates FAS
2) Extracellular ligands part of the TNF family e.g. TNF-alpha, TRAIL
3) Receptor activation leads to activation of the cytoplasmic Fas-associated death domain protein (FADD)
4) Initiates caspase cascade via caspase 8 activation

Question 17

Describe another extrinsic p53 regulated pathway of apoptosis

Answer 17

Insulin-like growth factor binding protein-3 (IGFBP-3):

1) p53 transcriptionally upregulates IGFBP-3 gene
2) Binds to extracellular IGF-1/2
3) Normally, IGF-1/2 bind to IGF-1/2 receptor on cell membrane and give pro-survival signals via the PI3 kinase pathway
4) Binding of IGFBP-3 to IGF-1/2 prevents binding of IGF-1/2 to receptor - induced apoptosis

Question 18

How do Nutlins target the p53 pathway as a cancer therapy?

Answer 18

• Bind to mdm2 and prevent p53 interaction
• Leads to stabilisation and activation of p53
• Cancer cells arrest in G1 or G2 and undergo apoptosis
• In phase 1 clinical trials
• Requires non mutant p53

Question 19

How do RITAs target the p53 pathway as a cancer therapy?

Answer 19

• Inhibit same interaction as Nutlins

- Binds to p53 instead of mdm2

Question 20

What treatments are available in patients with mutant p53?

Answer 20

Tumour-associated mutations destabalise p53 tetramer

Therapies aim to stabilise tetramer

PRIMA-1

• Prevents unfolding of mutant protein
• Maintains stability and activity of p53