Lecture 4 - Tumour Suppressor Genes - Mechanisms of Action

What are the three main cell cycle checkpoints?

G1/S checkpoint

G2/M checkpoint

Spindle (metaphase) checkpoint

What occurs during the G1/S checkpoint?

DNA replication is blocked if unrepaired DNA damage exists

• If the DNA damage is irreparable then the cell undergoes apoptosis

What occurs during the G2/M checkpoint?

Mitosis is blocked unless replication and repair of DNA is complete

What occurs during the Spindle (metaphase) checkpoint?

Metaphase is blocked until all the chromosomes are correctly attached to the spindles

E7 protein of HPV binds to what TS protein?

pRB

How does pRb become inactivated?

By phosphorylation. The more phosphorylation, the more inactivated it becomes 

In cancer, how can pRb become inactivated?

By a mutation or by viral oncoprotein binding

• This results in uncontrolled cell proliferation

What is the mechanism of normal pRb function?

• pRb starts hypophosphorylated in G1 phase

• Phosphorylation then increases as the cell progresses through the cell cycle

• Prior to DNA synthesis pRb is phosphorylated by CYCD1/CDK4 complex

• When phosphorylated, pRb releases from E2F/DP complex

• This results in transcription of genes which promote entry into S phase

What TS protein is CYCD1/CD4K inhibited by?

Inhibited by p16

When pRb is not phosphorylated, what happens?

pRb binds with E2F/DP complex which inhibits transcription.

• Does this by recruiting proteins involved in transcriptional repression 

• This prevents the cell from entering S phase, causing cell cycle arrest

How can pRb be inactivated in cancer and what does this cause?

Can be inactivated by mutations/viral oncoprotein binding

• This results in uncontrolled cell proliferation because pRb cannot prevent cell cycle entry

When pRb is phosphorylated, is it activated or inactivated?

Inactivated.

• Cell cycle can progress

Where on chromosome 17 is p53 gene found?

p region of chromosome 17

Vast majority of mutations in p53 are….. mutations.

Fill in the blank.

Vast majority of mutations in p53 are missense mutations

• Causes amino acid substitutions in the central portion of the protein 

What gene is mutated in Li-Fraumeni syndrome (LFS)?

One copy of p53.

• These families are at risk of developing many different cancers.

E6 protein of HPV binds to what TS protein?

p53 and inactivates it

p53 is inactivated by what normal cell protein?

MDM2

• Binds to p53 and hides its transcriptional activation domain

• Also promotes ubiquitination and destruction 

p14 (ARF) gene is involved in regulation of what protein complex?

p53/MDM2 complex regulation.

What is p53 and what functions does p53 have?

Transcription factor protein

• Enhances genes involved in cell cycle arrest and apoptosis 

• Controls cellular response to DNA damage

• Stalls cell cycle for DNA repair

Cellular p53 is usually very high in case the cell needs to undergo apoptosis.

True or False?

False.

Cellular p53 is very low as it’s rapidly degraded. 

What can increase p53 expression in the cell?

Cell stress/damage sensors can lead to the phosphorylation and stabilisation of p53

What are the three main domains of p53?

Central core domain

Amino terminal domain

Carboxy terminal domain

What is the role of the central core domain of p53?

Binds specific DNA sequences

What is the role of the amino terminal domain of p53?

Transcriptional activation domain

What is the role of the carboxy terminal domain of p53?

Self dimer/tetramerization domain

p53 activates transcription of genes which control:

• Cell cycle

• Apoptosis

• Self-regulation of p53

• DNA damage repair

p53 represses the transcription of genes like:

• Cell cycle promoters

• Apoptosis inhibitors

How does p53 self-regulate itself?

p53 enhances transcription of MDM2 which inhibits p53.

• This creates a negative feedback loop which regulates p53

How does p53 direct cell cycle arrest via p21?

Induces p21

• p21 then inactivates cyclin-dependent protein kinases which usually phosphorylate pRb

• This prevents pRb phosphorylation which prevents its inactivation

• This causes cell cycle arrest

Where on chromosome 9 is the INK4/ARF locus located?

Located on p region of chromosome 9

What are the three proteins encoded by the INK4/ARF locus?

p16 - Also called INK4a

p15 - Also called INK4b

p14ARF - Due to an Alternative Reading Frame

• Structurally unrelated to p16 and p15 

How does p16 regulate cell cycle?

Regulates pRb phosphorylation

• Inhibits CYCD1/CDK4 complex which phosphorylates pRb

In cancer, inactivation of p16 causes what?

Inappropriate phosphorylation of pRb and loss of G1/S checkpoint control 

How does p14ARF regulate the cell cycle/apoptosis?

Regulates through p53

• When proliferative signals exceed a threshold, ARF binds and destabilises MDM2

• Leads to a p53 response due to accumulation of p53 

In cancer, loss of p14ARF causes what?

Excess MDM2 and loss of p53-dependent control  

How does p14ARF protein differ from p16?

Contains sequences from a distinct first exon.

• Also has alternative exon usage and alternative reading frame to p16.

  • Hence p14ARF (Alternate Reading Frame)

Inactivation of only p53 or pRb is probably needed to avoid cell checkpoint and apoptosis.

True or False.

False.

It’s generally thought that inactivation of BOTH p53 and pRb needs to occur to avoid the cell checkpoint and apoptosis.