L10 + L11 - Cell Cycle Flashcards
4 causes of cancer
Enviro - carcinogens
Viral infection
Inherited factors
Genetic instability
Normal cells will only proliferate when …
There are growth factors present
Absence of growth factors =
No proliferation
What can other signals cause to happen to grwoth factors
Overrule the stimulatory effects of growth factors and force a halt to prolife
EC signals can induce a post mitotic differentiated state
What is significant about this state
There is no proliferation
What is the cell cycle clock
Master governer
Network of interacting proteins that recieves signals from the outside and inside - integrates these signals and then decides on the fate of the cells
cell proliferation is controlled by
Cooperation of a veriety of proteins
What are growth factors
Small proteins
Travel through EC space to covery messages to other cells
What are growth facotrs also known as
Mitogens
Why are growth factors often known as mitogens
Indicating their ability to induce proliferation
Uncontrolled cell proliferation can lead to
The formation of a tumour;
How do cancer proteins inflcuence the cell cycle clock
Disruption of the normal control mechanisms leading to sustained proliferation
Alternative ways cancer cells induce prolierations
Auto produce growth facotrs
Signals stimulate surrounding cells to cause production of GF
Degreulation of GF receptors (leads to seemingly inc conc of GF)
Consitit act of GF downstream targes
Disruption of negative feedback which atteuates signalling
G1
first gap pahase
Key decision to prolif or remain quiescent
8-10
G0
Non growting
Withdrawl from cell cycle
Is G0 reversible
Can be either
What cells is G0 irreversible
Neurones of the brain
How can G0 be revered
Through stimulation with mitogens
S
Replication
6-8 hrs
What cells is S phase faster
Normally proliferative cell types - lymphocytes
G2
Cell growth continues
Replication of the centrososmes
When is the decision to proliferate made
In G1
What is the difference between cell growth and cell division
Growth = accumulation of cellular constituents
Division = actual splitting of the cells via mtiosis and cytokinesis
What methods has histoirically been used to study the cell cycle
Flow cytometry
Advantages of flow cytometry
Fast
Quantitative
Flow cytometry analsyses …
DNA conent - cells must be loaded with a flourescent dye which lables the DNA
Describe what would be seen in flow cytometry of a cell in G2 or M phase
Would have double the DNA
What other methods may be used to study the cell cycle
Stages of mitosis
Immunoflourescence and epiflourescence microscopy
During G1 there is a discrete window to ….
This occurs …
Known as …
consult the EC environment
Onset of G1 to just before G2
Restriction R point
If GF and serum are removed before the cells have competed 80-90% of G1
Cells fail to proceed and exit into G0
If serum and GF removed in the final hour of G1
Cells proceed to S, G2 and M phase
If a cell goes past the restriction point what must it do
Complete the rest of the cell cycle
Why is there a discrete window to consult the EC environement
Downregulation of R point machinery in G1
What experiements were performed by Rao and Johnson
Nuclear fusion experiements
Mixing nuclei in the same cytoplasm to see if they influenced each other
Mixing multiple nuclei in the same cytoplasm is also know as `
Heterokaryon
S and G1 nuclei fused
Gives 2 s phase
S and G2 nucelus
No effect
G1 and G2 nucelus
No effect
Interphase and mitotic nucelus
Two mitotic nucelus
What conclusion can be made from the heterokaryon expt regarding:
THE S PHASE NUCELUS
Contains a diffusible factor inducing replication
What conclusion can be made from the heterokaryon expt regarding
G2 NUC
G2 nucelus is resisitant to the S phase promoting factor
What conclusion can be made from the heterokaryon expt regarding
G1 AND G2 NUCE
they have no effect on each other
What conclusion can be made from the heterokaryon expt regarding
Mitotic nuceli
Mitoitc nuceli release mitosis promoting factor that affects all interphase nuclei
two core components of the cell cycle clock
Cyclins and CDKs
CDKS are
Kinase which have multiple tatger
What is the effect of cyclins on CDKs
Activate the catalytic activity (can increase by up to 400’000 fold)
Help with substrate recognition of the cyclin CDK complexes in the cells
3 model systems for cell cycle study
yeast
Frog Eggs
Sea urchin embryos
Adavantage of frog eggs
Biochemical analysis is easy due to the size
Two cell cycles shown in Yeast
Fission and budding
What different mutatns were created in yeast
Wee
Cdc
Temperature sensitve
Describe the principles of a temperature sensitive mutation
At a permissive temperature the protein has wildtype function
What substance was discovered in frog eggs
Maturation promoting factor - AKA - mitosis promoting factor
What is MPF linked to
A protein with kinase activity which osciallates during the cell cycle
What happens if you were to put sea urchin embryos in soapy water
They start dividing
What was seen when proteins in the C urchin embryo were labelled with radioactive-methionine
Shows that one protein accumulates as cells get ready to divide
This then disappear when division occurs
Injection of cyclin is suffience to ….
Induce maturation and activation of MPF
What is the 2 major activites of MPF
Binding of cyclin to Cdc2 kinase
Phosphorylation of cdc2
What two enzymes act to regulate activity of CDKS
Wee 1 kinase
Cdc25 phosphatase
What cyclin/CDK pairing involved in
G1
CDk4 and CDK6 depend on the association with cyclin Ds
What are the D type cyclins
D1 D2 and D3
What cyclin/CDK pairing involved in
After the R point
E type cyclins associate with CDK2
Then phosphorylation of the substrates required for the entry into S phase
What cyclin/CDK pairing involved in
S
A type cyclins replace E type cyclins in complex with CDK2 this causes S phase progression
Later in S phase A type cyclins associate with CDC2 AKA CDK1
CDK1 AKA
CDC2
What cyclin/CDK pairing involved in
G2 phase
B type cyclins replace the A type cyclins in complex with CDC2
What cyclin/CDK pairing involved in
M phase
B type cyclins in CDC2 trigger the entry into mitosis
What cyclin/CDK pairing involved in
G0 to G1
Mediated by cyclin C in complex with CDK3
Describe the fluctuations of cyclin E during the cell cycle
Low levels throughout most of G1 then rapid increase after the R point
Describe the fluctuations of cyclin A during the cell cycle
Levels increase when the cell enters S phase
Descrube the fluctuations of cyclin B during the cell cyce
Levels increase in anticiptation of entry into M phase
Why does cyclin level decrease at various points in the cell cycle
Through degradation in a ubuiquitin dependent manner
What is the reason that the cell cycle can only proceed in one direction
The cycling levels of cyclins
Why is D cyclin an exception
Exp at constant levels and regulated by EC signals
What controls the expression of D type cyclins
Growth factors and integrin mediated ECM attachement
D type cyclins convey ..
Messages from the EC environment to the cell cycle clock in the nucleus
Where are D cyclins synthesised
In the cytoplasm and then transported to the nucelus
Removal of growth factors leads to
Rapid collapse of cyclin D1 levels
Describe the two possible fates of D type cyclin in G1 what is the effect that this would have on the cell cycle
If favourable environment then cyclin D enters the nucleus and the cell cycle goes on
If unfavourable environment then the cell cycle arrests in G1
D type cyclins respond to
EC environment
How do cyclins/CDKs help with the forward progression of the cell cycle
Activate in the next phase
Inhibit complexes involved in previous phases
What are CKIs
CDK inhibitors
What proteins inhibit D type cyclins
P16, P15, P18, P19
Why write out the inhibitors of D cyclins as
P16 P15 P18 P19
Because superscripts then go
p16 - INK4A
P15 - INK4B
P18 - INK4C
P19 - INK4D
What is the other group of CKIs that inhibit E-CDK2, A-CDK2, A-CDC2, B-CDC2
P57
P27
P21
At the start of G1 what is the cyclin CDK pairing?
D-CDK4/6
After the restriction point what happens to the cyclins and CDKs
Cyclin E is brought into a complex with CDK2 triggering S phase
What complex is responsible for driving the cycle into S phase
E-CDK2
What happens in S phase to the E-CDK2 complex
E cyclin replaced with A cyclin forming A-CDK2
CDK2 is then replaced with CDC2 forming A-CDC2
What is the complex is G2 phase
B-CDC2
B cyclin replaces the A cyclin
What cyclin/CDK complex is responsible for driving the cell into M phase
B-CDC2
What is the role of TGF-B in the early stages of carcinomas
TGF-B arrests the growth of many stages of cancer
What is the role of TGF-B in the later stages of carcinomas
Contributes to the invaseivness of tumours
What is the molecular mechanisms implicating TGF-B in the control of the cell cycle clock (R point)
TGF-B increases the levels of P15(INK4B) leading to inhibition of D-CDK4/6 so cells unable to make it to the cell cycle
What does TGF-B weakly induce?
What is the effect of this?
Increases the levels of P21(Cip1) cell cycles is then halted
What provides a stronger stimulation of P21(Cip1)
Why is this important
Stronger induction through DNA damage
Ensures the cell cycle does not progress into S phase and copy damaged DNA
Akt AKA
P-B
What is the action of Akt on P21
Phosphorylation in the nucleus causing translation to cytoplasma
What is the effect of Akt on P27
Phosphorylation in the cytosol blocking nuclear translocation
How may Akt be related to human tumours
Akt activation correlates with cytoplasmic P27 and doesnt block cell growth
What must happen to Akt (PK-B) for it to become active
What is the active version known as as a result of this
Must be first phosphorylated
Induces a confirmational change
Phospho-Akt
What is the result on P71 and the cell cycle of there being low levels of phospho-Akt present/
Localisaiton of P27 is strongly nuclear
Is able to act to block progression of the cell cycle
What is the effect of active PKB (Akt)
Activated through phosphorylation
P27 in the cytoplasm
Unable to block progression of the cell cycle
Is outlook better for patient when P27 is localised only to the nucleus or the cytosol
Better outlook when P27 only in nucleus - able to have more of an effect on arresting the cell cycle
Does P27 being located in the cytosol cause the cancer
NO
Correlation here and not causation …
