Lectures 3&4 - Cell cycle and cancer

Question 1

What are the cell cycle phases?

Answer 1

G1 – preparation for division (G0 is another stage that occurs when cells are not ready to replicate/dormant to replication)

S phase – synthesis - >2n – DNA is being replicated, growth of cell (not just replication of DNA but also organelles)

G2 – making checks that everything’s okay before mitosis

M - mitosis - division of the cell made up of several phases

Question 2

What are the phases of mitosis?

Answer 2

Interphase – everything other than mitosis

Prophase – DNA “untangled” and starting to condense

Prometaphase – microtubules linking to the spindle poles, attach to chromosomes and start to line up

Metaphase – sensors of tension across the centromere, if tensions even then they will send diffusible signals to cut, if not even then it will prevent cutting, microtubules pull apart equally from both sides

Anaphase

Telophase – fission of membranes

Cytokinesis – separation and generation of full new cells (with all organelles present

Question 3

Describe the usual states of cells/cell cycle phases in normal adults

Answer 3

Most cells in the body are in G0

-some of these can re-enter the cycle in response to stimuli (e.g. liver)
-others are terminally differentiated (e.g. neurons) - not many more formed after birth

Some tissues contain continuously dividing cells (stem cells) (e.g. gut, skin) - regenerate due to constant damage, as this may be easier than repair

Question 4

what are two “rules” of the cell cycle?

Answer 4

Alternation – cell cycle events always occur in the right order - e.g. mitosis to G1, can’t just go straight back to M

Completion – one process must finish before the next one starts

Question 5

Describe an early fusion experiment which gave insight into how the cell cycle works

Answer 5

1. Separate cells out based on their cell cycle phase
2. Polyethane glycol to combine/fuse cells to end up with cells with two nuclei
   Results:
   - If you fuse a mitiotic cell with any other cell, theres a signal from mitotic cell that causes the other cell to enter mitosis, shows that this phase is dominant over the other ones
   - Merge S cell with G1 cell you end up with S phase being dominant
   - Take a G2 cell and fuse with S phase cell, signal from S phase cell to G2 to cell that causes a delay, we don’t want the G2 cell to go to next stage without s phase cell having fully replicated DNA

Question 6

what is alteration of the cell cycle ensured by?

Answer 6

cyclins and cdk

Question 7

what are cyclins?

Answer 7

The regulatory subunits of cdk (cyclin-dependent kinases)

Question 8

Describe cdk regulation

Answer 8

Regulated by phosphorylation/by cyclin-dependent kinase inhibitors (cdkis)

Cyclin binding allows phosphorylation of cdk at T160 by cdk activating kinase (CAK)

Cdks are inhibited by phosphorylation at T14 and Y15 (by wee1 and mik1 kinases). These sites are de-phosphorylated by Cdc25, a promoter of mitosis

Cyclin binding changes conformation of cdk, allowing its phosphorylation by CAK on the T loop

Question 9

what are cyclin-dependent kinase inhibitors (cdkis)

Answer 9

inhibit cdk activity. Natural molecules but also therapeutic systhesised molecules can be cdkis

Question 10

Decribe the two main gene families of cdkis

Answer 10

CDKN2 gene family (A-D) (also called INK4 - in protein terms)
-Proteins p16INK4A, p15INK4B, p18INK4C, p19INK4D
-Number stands for size of protein when run on western blot
-Bind and inhibit cdk4 and cdk6

CDKN1 gene family (A-C) (also called Cip and Kip)
-Proteins p21Cip1/WAF1, p27Kip1, p57Kip2
-Inhibit E- and A- dependent cdks
-Point of action at the cyclin rather than kinase

Question 11

Explain the cyclin/cdk cycle

Answer 11

increase in activity of CDK4/6 (tend to vary between 4 and 6 depending on cell type) - increase due growth factors inducing cyclin D production which binds to CDK4/6 and causes them to be active

trigger to S phase comes from when cell starts making cyclin E which binds to CDK2, activating it

S and G2 phase, Cyclin A made instead of E, this also binds to CDK2 driving cell cycle to the end of G2

Sharp drop off of activity of CDK2 before Mitosis - activity switched to Cyclin B-CDK1

Cyclin B activates CDK1 - cdc25 (a phosphatase also removes inhibitors at this point)

Flip to anaphase due to loss of CDK1 activity

Question 12

explain how cdk1/cyclin B activates the anaphase promoting complex (APC)

Answer 12

production of cyclin B in between interphase and prophase leads to phosphorylation and activation of the protein cAPC - anaphase promoting complex

This activation of APC leads to polyubiquitination of the cyclin - which allows it to be targeted for degradation

This degradation allows for anaphase to occur due to loss of CDK1 activity caused by Cyclin B degradation

Tension signals also feed into APC system and shut it down (so that degradation doesn’t happen too fast)

Question 13

which checkpoint of the mitosis prevents aberrant mitosis

Answer 13

anaphase

Question 14

explain what happens at the G1 (R) checkpoint

Answer 14

1. Ras stimulates synthesis of cyclin D - binds to cdk4/6, once activated it moves into the nucleus and phosphorylate Rb
2. Rb on promoter region of gene - coupled through genes by transcription factors E2F and DP. These would usually drive forward the transcription of genes involved in S phase, but Rb is a transcription repressor, so shuts down this transcription
3. When phosphorylation occurs (by cdk4/6 and cyclin D) it partially reduces Rbs activity as a transcription repressor and allows transcription of some S phase genes e.g. cyclin E
4. Cyclin E gets made and binds cdk2 in the cell
5. Cdk2 is kept shut down by p27kip1 (it is a cdk inhibitor). So there still isn’t any active cdk2 yet
6. Eventually so much is made that you start to get some cyclin E-cdk2 action due to there being higher levels of this than of p27kip1
7. Cyclin E-cdk2 can phosphorylate p27Kip1 which causes attachment of skp2, skp 2 recruits SCF (a ubiquitin ligase complex) which causes breakdown of p27kip1 by ubiquination tag, degradation by proteosome
8. The remaining cyclin E-cdk2 complexes phosphorylate the remaining sites on Rb, fully deactivating it, so that E2F and DP proteins can drive the transcription of S phase genes, driving the cell into S phase

Question 15

what two of the 6 hallmarks of cancer that relate to the cell cycle

Answer 15

• self sufficiency to growth factors
  -insensitivity to anti-growth signals

Question 16

explain how a lot of cancer cells can pass through the G1 R-checkpoint

Answer 16

due to the absence of an external growth factor stimulus either due to:

Constitutively active growth factor signalling (always accelerating)
or
R-point defects (brake broken)

Question 17

what are the Oncogenic growth factor defects in cancer

Answer 17

Continuous exposure to growth factors

Over-expression of growth factor receptors

Activating mutations in growth factor receptors

Activating mutations in growth factor signalling molecules e.g. ras

Question 18

Explain details of the ErbB2/HER2 receptor

Answer 18

tyrosine kinase growth factor receptors

intracellular and extracellular parts, when extracellular receptor is bound it activates the kinase part in the intracellular side of the cell, this then phosphorylates tyrosines

Overexpressed in many cancers (especially breast) – due to gene amplification

15% of breast cancers are defined by this - treated with a therapeutic antibody that targets the receptor

Question 19

what is the Ligand for ErbB2?

Answer 19

heregulin/neuregulin

Question 20

what is the Ligand for ret?

Answer 20

GDNF (glial cell derived neurotrophic factor)

Question 21

Explain details of the Ret receptor

Answer 21

growth factor receptor – fusion translocations lead to cancer (NOT amplifications)
– gene gets rearranged so that either its next to a promoter for another gene that’s always on or fusion so that the protein itself is abnormal and you get the same amount of protein but its always active

Ret is a protein expressed in the thyroid

Activating point mutations in the receptor that can be inherited, can lead to:

Familial MEN – multiple endocrine neoplasia

Question 22

95% of pancreatic cancers have a mutation in which oncogene?

Answer 22

Ras

Question 23

name one of the Restriction point defects in cancers, and how it works

Answer 23

Cyclin D1 - Oncogene

Over-expression due to growth factor signalling (many cancers)

Cyclin D1 gene amplified (breast (13%), oesophageal (30%))

Cyclin D1 gene rearrangement. Mantle cell lymphoma (100% express cyclin D1)

Question 24

name another one of the Restriction point defects in cancers (not cyclin D1), and how it works

Answer 24

CDK4 & CDK6 – Oncogenes

On different chromosomes, expressed in different cell types

CDK4 different tumors will cause different things to the cell that lead to the same outcome

CDK6 different set of cancers, some translocations occurring

Question 25

name one other Restriction point defects in cancers (not cyclin D1 or CDK4/CDK6), and how it works

Answer 25

CDKN2A & CDKN2B – TSGs (p16INK4A, p14ARF & p15INK4B)

Proteins that inhibit cyclin dependent kinases – they are tumor suppressors as job is to keep cell cycle in check

INK4b feeds into the Rb part of the checkpoint as helps control cdk4 and cdk6

Mutations of exons and methylation of promotor of gene – both lead to INK4b not being expressed and cell cycle continuing

INK4a also feeds in to rb pathway

Question 26

name ANOTHER Restriction point defect in cancers (not cyclin D1,CDK4/CDK6 or CDKN2A & CDKN2B ), and how it works

Answer 26

Rb (TSG)

Gene lost or mutated in rare cancers – retinoblastoma, osteosarcoma, small cell lung cancer.

Both gene copies (alleles) must be lost to promote cancer

Question 27

Name the last Restriction point defect in cancers (not cyclin D1,CDK4/CDK6, CDKN2A & CDKN2B or Rb ), and how it works

Answer 27

CDKN1B (p27Kip1 (TSG) and SKP2 (oncogene))

Decreased p27Kip1 expression correlates with progression in many tumour types (e.g. colon, breast)

This may be due to increased levels of Skp2, e.g. in breast cancer (S.Signoretti et al, 2002)

Question 28

explain how the hallmark to cancer: Insensitivity to anti-growth signals, works overall

Answer 28

Cancers are also defective in other G1/S checkpoints:

Inhibitory growth factors (e.g. TGFβ)

DNA damage checkpoints (p53)

Question 29

Explain how mutations in TGFβ lead to cancer

Answer 29

shift the TGFβ response from growth inhibition to growth promotion:

Defective DNA repair (MSI) in 15% of colon cancers leads to inactivating mutations in TGFβRII

Other colon cancers loose the SMAD4 gene

Ras mutations can switch the cellular response to TGFβ

Question 30

Explain how the p53-dependent stress checkpoint works

Answer 30

P53 is a transcription factor

Constantly synthesised but can be rapidly degraded (15 minutes)

Constant feedback loop going on

Activated due to many different types of stress – DNA-damaging agents, hypoxia, oncogenic stimuli, ribonucleic depletion, senescence

Stress leads to transcription of a cdki:

p21/waf1 leading to cell-cycle arrest

or bbax which leads to apoptosis (only when very high stress)

Question 31

In what percentage of all cancers is the p53 gene mutated

Answer 31

50%

Question 32

Explain how p53 is a dominant negative tumour suppressor gene (TSG)

Answer 32

p53 is a transcription factor which binds DNA as a tetramer

One mutant p53 molecule in the tetramer inactivates the transcription factor

Therefore a mutation in one p53 allele in is sufficient to promote cancer

Question 33

Explain how Mdm2 is the major regulator of p53 activity in cells

Answer 33

Mdm2 is a ligase that directs the ubiquitination and degradation of p53

Oncogenic stimuli such as oncogenic ras, induce the expression of an Mdm2 inhibitor, p14-ARF

p14-ARF prevents ubiquitination of p53 by Mdm2

therefore p53 is not degraded

Question 34

P14-ARF is expressed from the same gene as..

Answer 34

p16INK4A

Question 35

what are the multiple mechanisms of p53 inactivation in human cancers

Answer 35

p53 point mutation and / or gene loss (TSG)

Loss of p14-ARF expression (TSG)

mdm2 gene amplification (Oncogene)