cell cycle in research and in the clinic

Question 1

what is G1 dominated by

Answer 1

G1 is dominated by the expression of cyclin D1 and the latter stages of G1 after the Restriction point we get cyclin e and then it gets replaced by cyclin a and cyclin b in G2 and Metaphase

Question 2

what is the major repressive unit stopping S phase from activating

Answer 2

phospho RB

Question 3

tumour suppressor

Answer 3

a tumor suppressor is something that when mutated or removed or made dysfunctional removes a natural break.

Question 4

what has the ability to be an oncogene

Answer 4

anything that is feeding into the central axis in a positive way has the ability to be an oncogene.

Question 5

tgf beta

Answer 5

tgf-beta is the ligand and tgf-beta receptor is where it signals through and then signals through the transcriptions smad 3/4 controlling the expression of p15.

Question 6

why do we want to monitor cell growth

Answer 6

when we’re in the lab we want to know how fast something is growing
- So it helps us to diagnose cancer. Look for abnormal growth.
- It helps us monitor how treatment responses are happening
- Lets us look for new drug treatments.
- develop personalised treatment plans

Question 7

cytostatic vs cytotoxic

Answer 7

cytostatic drugs cause the cells to stop growing
○ they cause an arrest in the cell cycle, but they are not necessarily removed
- Cytotoxics are causing cell cycle exit probably activating apoptosis or necrosis or some cell death
- and the difference is cytotoxic Drugs will make a tumors shrink and cytostatic will mean that it doesn’t grow so you don’t get progressive disease, but nor does it diminish?

Question 8

simply quantify cell number

Answer 8

• can count manually with a hemoctometer
• can stain the cells (crystal violet) quantify after with solubility
• BUT numbers is not cell cycle
• cell number is increased by proliferation but lost due to decreased adhesion or lower survival

Question 9

FACs and cell cycle

Answer 9

• fluorescent probes measure DNA in a stoichiometric manner
• probe molecules bound to DNA is proportional to the number of chromosomes
• diploid cells, cells synthesising DNA and cells pre-mitosis
• propidium iodide intercalates between DNA bases
• DAPI is bound to dsDNA in AT clusters

Question 10

what else do we have to make sure we do when we do this analysis

Answer 10

one of the other things that we have to make sure we do when we do this analysis
is to remove the RNA.
- if we’re staining DNA almost all of these stains are also staining RNA.
- So when we do this analysis, we have to make sure we had RNAse to get rid of all
the RNA before we do the staining

Question 11

if I was to inhibit receptor tyrosine kinases What would you expect to happen?

Answer 11

if we inhibit rtk signaling it is like removing a go signal which means all the cells will get stuck in G1 and they will not progress into s phase - will have this block at the end of G1.

Question 12

what does paclitaxel do

Answer 12

• inhibits microtubules
• it stops chromosomal segregation

Question 13

paclitaxel inhibits microtubules. It stops chromosomal segregation. So where would we expect cells to be?

Answer 13

if we stop cells in undergoing mitosis. We stop mitosis happening, the cells get blocked in G2, and they will no longer be able to progress. So you’ll end up with a blockage here.

Question 14

what does palbociclib inhibit

Answer 14

CDK 4/6

So if I’m inhibiting cdk4 and 6 right here before the Restriction Point you don’t
have activation of cdk 4 and 6 - I don’t get those initial phosphorylation events
on RB
- without those phosphorylations we don’t get the accumulation of cyclin e
and without cyclin e and cdk2 we do not exit G1.

Question 15

what does flavopiridol inhibit

Answer 15

CDK2, 4, 6 and CDC2

Question 16

does inhibiting CDKs cause cell death

Answer 16

what normally happens is a cell will be able to survive in one phase of the cell cycle for a particular amount of time, but then it will have to make State decisions Eventually.
It will have to decide whether it’s going to be able to survive in situ or whether it will have to undergo apoptosis or become senescence

Question 17

types of drugs that have been developed to inhibit the progress of cells from one stage to another

Answer 17

• so in G1, we’ve got hormonal drugs. We’ve got mitogen signaling Inhibitors
• in S phase. We’ve got things that interfere with DNA replication and DNA synthesis.
• We’ve got things that are acting in G2 - So these are the sorts of things that are inhibiting microtubule formation, interfering with the centrosomes
• and then in M phase we’ve gone things that interfere with the chromosomes splitting.

Question 18

oestrogen receptor in breast cancer

Answer 18

Oestrogen binds to the oestrogen receptor - in breast cancer This is really important.
- binding to the oestrogen receptor, activates transcription and leads to cell cycle progression.
- But if we use tamoxifen we can block this binding, but if we combine that with for example Cdk4 6 inhibitor we can decrease the cell cycle as well as decreasing the transcriptional output of these oestrogen receptor and therefore Target the cell in two different ways.

.

Question 19

when do we use inhibitors

Answer 19

we don’t really use any of these Inhibitors that act on CDKs in any single treatment as just stopping
cancer from growing isn’t really enough.
- But what we do is we use them in combination with something else.

Question 20

what can we do to look at whether cells are undergoing the cell cycle

Answer 20

one of the other things that we can do to look at whether cells are undergoing the cell
cycle is to look at specifically s-phase.
- So what we have here is brdu, so this is a fluorescent dye that can be Be incorporated into synthesizing DNA.
- So when cells are entering s phase brdu gets Incorporated along with normal nucleotides and then you can end up with fluorescent DNA
- and by doing this and labeling the cells, if we then look at the cells that I’ve got something that’s fluorescent green We can see the cells that entered S phase within the last hour or so.

Question 21

DNA staining cell cycle

Answer 21

• FACs see all phases of cell cycle
• BrdU or EdU only S phase
• both highly quantitative
• both require the cells to be fixed

Question 22

fucci cells

Answer 22

• stable or transient expression
• uses the protein stability changes for cell cycle dependent proteins
• can be used live or snapshots
• shows G1/0, S and G2M

Question 23

FUCCI CELLS

Answer 23

• live cell imaging
• complex model compatible
• hard to quantify unless with FACs
• not all cells stain not compatible with human tumour samples
• need cell lines that actively divide well

Question 24

Ki67

Answer 24

• a protein, that is a marker of cell proliferation
• the Ki67 protein is expressed during all active phases of the cell cycle (G1, S, G2 and M), not (G0) cells. it is involved in various processes related to cell division, such as chromosome condensation
• snapshot, but good for histology and patient samples