Mitosis checkpoints

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Why do we need checkpoints in mitosis?

Make sure DNA replicates completely & only once

Makes sure DNA is intact before mitosis begins

Make sure all chromosomes separate equally

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Key players in checkpoints

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Cell cycle signals

i) Internal signals

Cyclin & cyclin-dependant kinases

Move the cell from one phase to the next

Cause chromosomes to condense

Nuclear envelope to break down

Formation of mitotic spindle

CDKs = enzymes

Generally constant levels throughout cell cycle

Become active only when bound cyclin protein

Activate/deactivate other cellular proteins by phosphorylation

Cyclin = co-factors of CDKs

Levels 'cycle'

Synthesised & degraded @ appropriate times throughout cycle

Real regulators of cycle! B)

ii) External signals

Growth factors

Platelet-derived growth factor - fibroblast division in healing

EPO

Lack of nutrients = inhibition of cell division

Density-dependent inhibition​​​​​​

​​​​​​​See image below

Anchorage-dependent inhibition

​​​​​​​Cells must attach to a surface

Signalled to cell-cycle control system through membrane proteins + parts cytoskeleton

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G1/S checkpoint

Damage to chromosomal DNA is repaired & cell checks if environment is favourable for going into S phase

3 proteins play key role in controlling progression from G1 into S

pRB

E2F

p53

All above = tumour supressors!!

Mutations in genes which code for these proteins = common cause of cancer

They are all products of normal alleles located on retino-blastoma gene

What happens at the G1/S checkpoint?

2-4 hours before entering S phase, pRB is phosphorylated

By D cyclins + CDK4 &/or CDK6

Will remain phosphorylated until telophase

This releases E2F

E2F promotes transcription mRNAs needed to progress to next stage - notably cyclin A & cyclin E

Mutations to retino-b gene can lead to inappropriate E2F release!

If unrepaired DNA damage detected in cell - passage into S phase is blocked!

pRB & p53 act together to cause cell-cycle arrest @ G1 block before DNA replication can occur

If damage is un-repairable - apoptosis occurs

What does p53 do?

Stops cell cycle

Activates DNA repair enzymes

Irreparable damage = apoptosis

How does the cell control p53 (i.e. prevent it from unnecessarily causing cell-cycle arrest?)

p53 conc. = generally low

MDM2 ensures this by attaching ubiquitin to free unactive p53

This labels it for destruction (think beta-catenin in WNT signalling!)

Signals from DNA damage sensors causes phosphorylation of p53 which activates it

p53 acts to prevent CDK2 expression & thus block cell cycle

(**See mol med - DDR & cancer lecture pg.3 iii))

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G2/M checkpoint

Cell makes sure it's correct size & that DNA repair + replication have occurred during G2

CDK1 - cyclin B must be activated by Cdc25c phosphatase

Incomplete DNA replication + damaged DNA cause inhibition of Cdc25C

Prevents CDK1 activation

Cell size = indicator of available resources

Small size = bad sign

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M phase checkpoint

Cell checks whether all chromosomes are attached to tubulin spindles

Seperation of chromatids = anaphase

Triggered by 'cyclosome' (anaphase promoting complex)

Marks specific cell cycle proteins for destruction

i.e. cyclin A & cyclin B

Proteins that hold sister chromatids together

Kinetochores not attached to spindle microtubules secrete cyclosome inhibitor

If this is defective, chromatids may seperate prematurely

In order to proceed to telophase:

Kinetochores must be attached to chromatids @ centromere

Must be attached @ both sides - bipolar

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Checkpoints & cancer

How does all this relate to cancer?

Cancer = loss of control of mitotic division

More specifically, it is a loss of control of mitotic checkpoint control

Cancer is able to overcome the restrictions of checkpoints

Notably by inhibiting p53 activity

p53 is very important in stopping DNA damage cells from dividing

Has the power to force cells into G0 / keep in G1 arrest

Stimulates DNA repair enzymes

Can cause apoptosis if required

Targeting CDKs in cancer treatment

Attractive targets, but no working drug has been developed yet

Problems have included lack of specificity, redundancy & unexpected side effects