cell cycle

Question 1

signalling in cells

Answer 1

• loads of ligands
• lots of receptors
• a few kinases
• a few transcription factors
• lots of gene targets
• lots of phosphorylation targets

Question 2

tissue homeostasis

Answer 2

normally in a tissue you have something called tissue homeostasis and this is telling your organs how many cells there should be
- and to do this we normally have a normal bunch of stem cells that are the Self replicating forms of cells.
- Some of those will then go off to differentiate
- once their differentiated This is what causes tissue.
- So although it is not a strict dichotomy The proliferation side of a cell is normally the undifferentiated.
- So the undifferentiated stem cell is the type that will replicate and proliferate and then the differentiated cell is the one that will maintain and stay there normally until it dies.

Question 3

what do cancer cells normally do

Answer 3

ancer cells normally go from this differentiated state and then they move to a proliferative state. and this proliferated state is when they start actively engaging in the cell cycle.
- It also means that they generally use these differentiated markers.

Question 4

advantages of being in an undiferrentiated state for cancer cells

Answer 4

first off they can start proliferating so they can self-renew like stem cells.
○ So they’re able to divide rapidly
- they become resistant to apoptosis because once they are undifferentiated you normally can express multiple different types of receptor at the same time rather than just the receptors that are normally held on the differentiator population.
- It helps with Invasion and metastasis
- You have increased plasticity - they are more adaptable
- Decreased immune surveillance -reduced expression of surface markers
- Metabolic plasticity - altered metabolism

Question 5

the cell cycle

Answer 5

we have the beginning of the cell cycle here normally determined at the beginning of G1
- . The cell grows a little bit. It gets to a decision point
- replicates its genome in S phase
- then increases its number of organelles and things in G2 and then eventually will divide during mitosis.

Question 6

how do we control the cell cycle

Answer 6

• The cells are not really intelligent, but they do have basic logic. So they’re going to weigh
  up different factors and come to a conclusion about whether it’s good to divide or bad to divide and
  
  • some of those things that go into this will be differentiation factors. So are there enough differentiation factors telling me that I shouldn’t divide and I should just become whatever sort of cell is that I should become.
    ○ are there enough growth factors here telling me that I should divide
    ○ nutrients - Is there enough oxygen are amino acids available to me right now to get me through the cell cycle.
• So when it weighs up these decisions, it will determine whether it does one of two things
  ○ it will either differentiate being survival or migration
  ○ or it can go into DNA replication and then cell division.

Question 7

cyclins controlling the cell cycle

Answer 7

So the cell cycle itself is controlled by the fluctuation of cyclins
- different cyclins are dominant at different stages
- cyclin b Reaches its peak the end of mitosis before decreasing rapidly
- cyclin d is maintained a relatively high level for g1 until S phase begins
- cyclin e seems to be switched on at the end of G1 reaching its peak just before S phase and then fading
and then cyclin a is the cyclin that is mainly dominantly expressed during S phase.

Question 8

how are the cyclins coordinating

Answer 8

• they are controlling the cell cycle by binding to cyclin-dependent kinases
• So the CDKs are normally stably expressed within the cell but they form complexes with the cyclins and when they’re with their correct cyclin they become active.
• only when they’re in complex Are they active and once they are active they can phosphorylate
  
  • And it is the switching out of these Cyclins with CDKs that controls the cell cycle

Question 9

cycle of CDKs and cyclins

Answer 9

So at the beginning of G1, we have this situation here where cyclin D is bound to cdk4 and 6
- at some point It stimulates the expression of cyclin e
- Cyclin e will then bind to cdk2.
- This will then lead to the expression or cyclin a
Which will then switch out with e to cdk2 which then causes the expression of this cyclin-dependent kinase Cdc2. To so then switch out with a and then enter into G2 where cyclin B will be expressed and then switch out again

So this cycle of cyclins moving in and out of complex with the CDKs is what drives and determines the phases of the cell cycle.

Question 10

what drives the cell cycle

Answer 10

• active cyclin/ CDK complexes drive the cell cycle forward
• CDK expression is stable throughout the cell cycle
• cycles of transcription and proteolysis of cyclins controls cell cycle

Question 11

when are your cells responsive to growth factors

Answer 11

• your cells are only responsive to growth factors during growth Phase 1.
• This is when they express their receptors that will get stimulated by EgF or hgf.
• most of those growth Signals can only influence our behaviour during G1. And so this is where the Restriction point is found.
• It is near the end of G1 and at that point the cell stops responding to growth factors because it’s already pretty much committed to going into cell cycle and proliferation.

Question 12

why is there a restriction point at the end of G1

Answer 12

• the main reason why you have this restriction point at the end of G one is because the next phase s-phase is metabolically the most energy requiring
• so to duplicate your whole genome that cost. an awful lot of energy and it is that metabolic demand of S phase that the cells are weighing up at this restriction point

Question 13

what happens if you end up stuck in G2

Answer 13

If you end up stuck in G2 for too long you end up normally with an unsustainable genotype
- having double The amount of chromosomes leads to genotoxicity.
- you cannot stay long in G2 because you’ve got too many copies of genes that are detrimental to the cell.

Question 14

what stage is the most stable part of growth

Answer 14

the most stable part of growth is G1 where a cell can stay in G1 for most of the time.

Question 15

if we don’t get the production of cyclin e and the activation of cdk2 complex what can happen to the cell?

Answer 15

-the cell can choose not to enter active cell cycle. It can exit the cell cycle
○ now, there are lots of different determinations of what we can call this state.
§ So some people would call it quiescent - So this is when a cell is quiet
§ some will call it senescence and this is a slightly different form where the cell basically stays there and it can age.
§ Or some people just call it exited.

Question 16

what activates the cyclin D promoter

Answer 16

multiple transcription factors activate the cyclin D promoter from different receptors and ligands

Question 17

what leads to activation of motor

Answer 17

• So if we have activated ras we can activate pi3 kinase which can activate AKT which is going to eventually lead to the activation of mtor

Question 18

what is mtor

Answer 18

• mtor is a massive complex and the most important thing about mtor is it recognizes nutrient status
  ○ So it doesn’t just integrate this signaling pathway Downstream of ras it also integrates nutrient signals.
• when mtor is happily activated you can have activation of phospho S6 which controls ribosomes as well as elongation factor 4E, which controls the ability of cyclin D1 mRNA to become cyclin D1 protein.

Question 19

3 levels of cyclin D1 being controlled

Answer 19

Downstream of our receptor tyrosin, kinases we have the three levels of cyclin D1 being controlled.
○ It’s mRNA levels
○ its ability to get translated from mRNA into protein.
○ And then the amount of protein that there is altogether by controlling its proteolysis.

Question 20

what happens once we have active complex of CDK46

Answer 20

• once cyclib D1 has increased enough we will have this active complex of cdk four and six
• once we have active cdk4 and six it will phosphorylate a protein called RB
• when RB is a phosphorylated it no longer represses the genes required to activate S phase

Question 21

how does RB repress genes required for S phase

Answer 21

○ It has passive repression where it just occupies different sites. - So it is binding to these two different types of transcription factors here But because it is associated with them It’s not recruiting the transcriptional Machinery to start transcription.

○ It also has active repression. So this is where RB is sat there with these transcription factors and it Associates with histone deacetylases, Changes the histone status, closes up the chromosomes and stops transcription from being able to begin because the factors are not able to access it.

Question 22

what happens once CDK 46 is activated

Answer 22

• Once CDk 4 & 6 is activated. So the signaling has been integrated and got lots of cyclin d, cyclin d is binding to cdk4 and six, it Can then phosphorylate RB
• once RB has been phosphorylated it is no longer able to associate with the transcription factors e2F
• at this point its repression is stopped and we have activation of new genes.
• One of these genes is cyclin e
• Cyclin e will then bind to cdk2 and then we have now a second complex
• This second complex also phosphorylates RB.
• So this is a form of feed-forward signaling. So once one thing happens, it can increase the ability or something else to happen.

Question 23

levels at the beginning of G1

Answer 23

at the beginning of G1 there is very low levels of phospho RB and it gets phosphorylated at multiple different sites.
- Once we get to the Restriction point there is now activation of cdk 46 and cdk2 and this leads to hyperphosphorylation, releasing lots of the transcription factors which lead to the production of S phase related genes.

Question 24

S Phase

Answer 24

We enter into S phase, these transcription factors then get degraded but RB stays hyper phosphorylated.

Question 25

phospho RB phosphorylation in G1

Answer 25

during most of G1 phospho RB is barely phosphorylated
- it slowly accumulates as cyclin D expression is increased Downstream of growth factor signaling
- as this complex becomes more active. It gets phosphorylated
- at some point It reaches the critical signaling, this feed forward mechanism of cyclin E production starts.
- We end up with lots of cdk2 being present which then also phosphorylates phospho RB.
And then we end up with this hyperphosphorylated state all the way through until the end of mitosis.

Question 26

how can we control the cell cycle if we have only got this feed forward mechanism?

Answer 26

• So where are the brakes other than this restriction point?
• So the brakes come in the flavors of cyclin dependent kinase Inhibitor
• there are different families of them But they repress the signaling of cyclin dependent kinases
• So the rest of the cell cycle are coordinated with these Inhibitors.
  So the expression and the presence of these Inhibitors reduce the ability of cdk 46 or the other complexes from getting past their respective restriction points.

Question 27

what controls these cyclin dependent inhibitors

Answer 27

• these are controlled by different factors - so growth suppressors
• So tgf-beta can increase the expression
• if we have DNA damage so p53 for example - p53 activation can cause the increase in p21. For example,
• we have oncogenes that have both the ability to increase cell cycle but they can also have the ability to decrease cell cycle.
• So oncogenes don’t necessarily just have one function they can also produce cyclin-dependent kinase inhibitors.

Question 28

feed forward mechanism

Answer 28

We have some activation of the cdk4 complex
- This phosphorylates RB slightly as repression stops. We end up with more cyclin e production, more cdk2 activation and hyper phosphorylation of RB

Question 29

cyclin e in breast cancer

Answer 29

• if you have low levels of cyclin e you are more likely to survive breast cancer
• if you have high levels of cyclin e, however, you are far more likely to die in an earlier period of time
  ○ so you’ll have a a far more aggressive form of breast cancer if you lose some of these controls Or if you have an inability to control them,
• so if you have high levels of cyclin e all the time this feed forward mechanism can’t be switched off because we have hyper phosphorylation of RB all the time, loads of e2f activity lots of S phase.

Question 30

what happens if we have got lots of cyclin E - genomic instability

Answer 30

if we’ve got loads of cyclin e we can see that the chromosomes are not really aligned and have also now got all these different centrosomes.
- So the chromosomes aren’t just in the wrong order and not paired but their also being pulled into different areas instead of just two cells.
○ They’re forming perhaps maybe three or four or five different cells
○ and this genetic instability and this rearranging is beneficial to cancer because if they continue to do this, they’ll have a mixture of of different genetic backgrounds
○ . So those polyclonal tumors again will end up with a big mix of different genetic background and have the diversity to respond to treatments and You have the ability to adapt to the nutritional status and to adapt to new environments when we metastasize.

Question 31

cell cycle checkpoints

Answer 31

there are four
- in reality, There are only really 3 - The checkpoint described in G2 is really quite basic
it can hold cells in G2 But the chances are your cells will divide eventually it’s just a matter of time.○ It can’t hold them there because having double the amount of genes is toxic and your cells will either die or they will just try and divide.
- So the three main checkpoints
○ we got G1. It’s checking for DNA damage. It’s looking for the nutrient status
○ S phase- It’s also DNA damage checkpoint. It’s looking at DNA replication or is the whole genome damaged,
○ M phase - looking at the mitotic spindle and looking at whether the chromatids are correctly aligned.

Question 32

what can happen at checkpoints

Answer 32

f there is some error or damage - something that wants the cells to stop, it will reach the checkpoint and it will start to delay.
§ So there’ll be increased expression of those inhibitory factors p27 p21.
§ Those inhibitory factors will delay those cyclin-dependent kinases and then the cell can either repair the damage
- if it repairs the damage then the checkpoint can be qualified and the cell can re-enter the cell cycle.

Question 33

what happens if the delay at the checkpoint is long enough

Answer 33

• If however the delay is long enough, There is still 3 different possible outcomes
• one it can continue the cell cycle Anyway, this happens quite a lot in cancer
• or it can enter apoptosis. So it can have programmed cell death. - The cells can be eliminated. And they can know that they’re not going to make it through cell cycle. They’ll be accumulation of death signals and death signals will overcome them and apoptosis will happen
  □ or it can exit the cell cycle all together and become senescent or quiescent

Question 34

during G1 when we have receptor signaling, how can we have both inhibitory
and growth signals integrating at the same time?

Answer 34

the way in which this can work is by them controlling the different parts of the cell cycle Machinery.

Question 35

tgf beta - growth inhibitory ligand

Answer 35

• tgf-beta binds to its receptor and it activates the transcription of the inhibitory protein p15.
  ○ p15 is one of the inhibitory factors for the cdk4 6 complex, so Whilst this is being activated, We have more of this- it inhibits this signaling, It’s less phosphorylation of RB and cells don’t progress into S phase.
• It also has the ability to increase the expression of p21
  ○ p21 inhibits cdk2 complexes and CDC 2
  ○ so the rest of this cell cycle complex is also been inhibited.

Question 36

what controls whether cells are going to transition into S phase

Answer 36

The amount of phospho RB controls whether cells are going to transition into S phase.
- If you reach s phase cells are going to divide
- So these two main cdk complexes eventually controlling how much phospho RB there is
- and so we have go signals and we have stop signals
○ and mutations in most of these can be found in cancer.
○ So if you have an activating mutation in a go signal, that would be an oncogene.
○ If you have a deactivating mutation in a stop signal you’ll end up with getting rid of a tumor suppressor.

Question 37

cancer as a multi step process

Answer 37

• It requires the removal of several different factors
  ○ the generation or the evolution of a cell to gain the characteristics of being able to form a tumor.
  ○ So an example of this is we can remove a negative regulator of the cell cycle but we may also need to add the activity of a positive regulator and then on top of that we need to inhibit cell death
  and as we accumulate the mutations in genes we end up eventually to a cancer cell, which is transformed.