1 - CELLULAR GROWTH REGULATION Flashcards
What are the 3 general considerations for cell growth 3
o Growth of a population of cells
o Growth at the cellular level (the cell cycle)
o Loss of cells by programmed cell death (apoptosis)
3 general considerations for cell growth - Growth of a population of cells 2
– Distinguish between increase in cell numbers (hyperplasia) and increase in cell size (hypertrophy)
– Depends on integration of intra- and extracellular signals (checks on cellular physiology, growth and inhibitory factors, cell adhesion etc.)
3 general considerations for cell growth - Growth at the cellular level (the cell cycle) 3
– Cell growth = increase in size (sometimes growth refers to this only) and cell division
– Cell cycle phases (G1, S, G2, and M) G1 is growth phase 1, S is synthesis (where DNA is replicated), M is for mitosis
– Progression controlled at three key checkpoints (restriction points)
3 general considerations for cell growth - Loss of cells by programmed cell death (apoptosis) 3
– A coordinated program of cell dismantling ending in phagocytosis. Distinct from necrosis
– Occurs during normal development (e.g. separation of the digits, involution, immune and nervous system development)
– And in response to DNA damage and viral infection
What is a broad term used to describe Growth factors, cytokines and interleukins 1
Proteins
3 things that Growth factors, cytokines and interleukins do?
– Stimulate proliferation (called mitogens) and maintain survival
– Stimulate differentiation and inhibit proliferation e.g. TGF Beta (transforming growth factor)
– Induce apoptosis e.g. TNFα and other members of the TNF family (tumour necrosis factor)
3 things that Growth factors, cytokines and interleukins do - an example of stimulating proliferation 2
• Usually named after originally identified target e.g. EGF, FGF, Interleukins (IL2 & IL4), NGF •
But see also PDGF (platelet-derived GF) and IGF1 (Insulin-like GF – the main effector of pituitary growth hormone)
3 things that Growth factors, cytokines and interleukins do - an example of stimulating differentiation and inhibit proliferation 1
e.g. TGF Beta (transforming growth factor)
3 things that Growth factors, cytokines and interleukins do - induce apoptosis 1
e.g. TNFα and other members of the TNF family (tumour necrosis factor)
What are the three broad classes of growth factors, cytokines and interleukins 3
PARACRINE: produced locally to stimulate proliferation of a different cell type that has the appropriate cell surface receptor
AUTOCRINE: produced by a cell that also expresses the appropriate cell surface receptor
ENDOCRINE: like conventional hormones, released systemically for distant effect
Define Paracrine 1
PARACRINE: produced locally to stimulate proliferation of a different cell type that has the appropriate cell surface receptor
Define Autocrine 1
AUTOCRINE: produced by a cell that also expresses the appropriate cell surface receptor
Define Endocrine 1
ENDOCRINE: like conventional hormones, released systemically for distant effects
What happens when you add growth factor to cells? 2
They will respond and enter the cell cycle, start dividing
What is PDGF 1
Platelet-derived growth factor (PDGF) is a mitogen for certain cell types, especially cells of connective tissue and cells of the developing nervous system
What happens when PDGF is no longer available to cells
If the PDGF is no longer available, there is a plateau until they receive more PDGF
What happens if you add TGF Beta to cells 1
If the cells receive TGF Beta, they will stop dividing
What happens if you add TNF Alpha to cells 2
TNF Alpha will lead to the cells committing suicide and the number of cells going down
Name the phases of the cell cycle 5
G0, G1 , G2 , S , M
Cell cycle - sheet
On sheet
Mitosis is the….
separation of the chromosomes and the physical separation of the cell into 2 daughter cells
What happens after the M phase 1
After the M phase, one of the daughter cells undergoes interphase. The cell grows using nutrients to create more cytoskeleton etc.
How do cells grow after the M phase 1
After the M phase, one of the daughter cells undergoes interphase. The cell grows using nutrients to create more cytoskeleton etc.
What happens in the growth phases 1
In the growth phases the cells get ready, so they have everything that is required for mitosis
What are Quiescent Cells 1
Quiescent Cells are cells that are arrested in the G0 phase
Define Quiescence 1
Quiescence is the reversible state of a cell in which it does not divide but retains the ability to re-enter cell proliferation. Some adult stem cells are maintained in a quiescent state and can be rapidly activated when stimulated, for example by injury to the tissue in which they reside.
Pathways for Quiescence cells 3
Re-enter the cell cycle and start proliferating
Terminal differentiation
Apoptosis
How can Quiescence cells re-enter the cell cycle 1
These cells can re-enter the cell cycle if we add mitogens and will start proliferating
What cells undergo terminal differentiation 1
Some quiescent cells may start to differentiate. E.g. they could become gut cells. This is called terminal differentiation. Some cells undergo apoptosis and die
How many copies of each chromosome are there after cell division 1
After cell division, the cells have two copies of each chromosome so that is 2N
How many copies of each chromosome are the S phase 1
After the S phase, there is more duplication, so it is 4N
Name one technique where you can find out the DNA content of a cell 1
You can use a fluorescence-activated cell sorter (FACS) to analyse the cell DNA content.
How does FACS analyse cell DNA content - steps 3
- Take cells and label the DNA with dye.
- The dye is read by a laser and the laser tells us how intense the cells are in each phase.
- We can see in what stage cells are in.
What does this graph show? 1 What difference can you see in the S phase? 1
o This graph shows the difference between a slow and fast rate of proliferation.
o In the high rate of proliferation, there are a lot more cells that are in the S phase
DNA replication - steps 4
1) DNA is replicated semiconservatively (daughter cells inherit one parental and one new strand).
2) New DNA is synthesized in the 5’ to 3’ direction from deoxynucleotide triphosphate precursors at a replication fork by a multienzyme complex (a replication machine).
3) Fidelity is determined by base pairing (A=T, G≡C) and presence of a proof reading enzyme in DNA polymerase.
4) Synthesis of the new DNA strand uses an RNA primer and occurs continuously on the leading strand and discontinuously on the trailing strand (giving rise to Okazaki fragments, which are ligated together after removal of the RNA primer).
Mitosis stains - diagram
– Stain in blue is for DNA
– Stain in red is for gamma tubulin which is required to form the microtubules that will bind to the centrioles and chromosomes
– Stain in green is for CHEK2 which is a cell cycle checkpoint protein
Name the four main stages of mitosis 4
Prophase
Metaphase
Anaphase
Telophase
Mitosis - Prophase 3
– Nucleus becomes less definite
– Microtubular spindle apparatus assembles
– Centrioles (yellow) migrate to poles
This is due to the overlap in the green and the red
Mitosis - Prometaphase 2
o Prometaphase
– Nuclear membrane breaks down
– Kinetochores attach to spindle in nuclear region
Mitosis - Metaphase 1
o Metaphase
– Chromosomes (blue) align in equatorial plane
Mitosis - Anaphase 1
o Anaphase
– Chromatids separate and migrate to opposite poles
Mitosis - Telophase 1
o Telophase
– Daughter nuclei form
What stage happens after mitosis? 1 Describe it? 2
o Cytokinesis
– Division of cytoplasm
– Chromosomes decondense
What types of drugs act on the cell cycle? 2
S-Phase active
M-Phase active
S-Phase active drugs examples - cell cycle 3
Fluorouracil
Bromodeoxyuridine
Tamoxifen
M-Phase active drugs examples - cell cycle 3
Colchicine
Vinca alkaloids
Paclitaxel
S-Phase active drugs - Fluorouracil
Fluorouracil (an analogue of thymidine blocks thymidylate synthesis).
So, DNA can’t make 2 copies (duplicate), so arrested in s phase of cell cycle.
S-Phase active drugs - Bromodeoxyuridine
Bromodeoxyuridine (another analogue that may be incorporated into DNA and detected by antibodies to identify cells that have passed through the S-phase).
S-Phase active drugs - Tamoxifen
Tamoxifen is an antagonist of oestrogen, stops cell growth. We know that breast cells need oestrogen to work so we can use it to decrease proliferation in ER-positive breast cancer cells
M-Phase active drugs - Colchicine
Colchicine (stabilises free tubulin, preventing microtubule polymerisation and arresting cells in mitosis – used in karyotype analysis), so chromosomes cannot separate, arrest at M phase.
M-Phase active drugs - Vinca alkaloids
Vinca alkaloids (similar action to colchicine)
M-Phase active drugs - Paclitaxel
Paclitaxel (Taxol, stabilises microtubules, preventing de-polymerisation)
What disease are 5-Fluorouracil, paclitaxel, the vinca alkaloids and tamoxifen used to treat? 1,1
5-Fluorouracil, paclitaxel, the vinca alkaloids and tamoxifen are used in treatment of cancer.
The idea is to stop cancer cells dividing.
What do the Cell cycle checkpoint involve 1
Controls (involving specific protein kinases and phosphatases) ensure the strict alternation of mitosis and DNA replication.
What do Protein kinase do 1
Protein kinase – regulates by phosphates do the opposite job, add phosphate groups to substrates
What is the only phase where cells are responsive to growth factors 1
G1 is the only phase where cells are responsive to growth factors
What are the 3 cell cycle checkpoints 3
– Before the S phase
– Before the cells start Mitosis
– Another checkpoint in mitosis
What happens in the cell cycle checkpoint - Before the S phase
o Checks the cells size, the nutrients, can the cell proceed by expending so much energy?, make sure that the DNA is not damaged
What happens in the cell cycle checkpoint - Before the cells start Mitosis 2
o Is the DNA completely replicated?
o Is the DNA not damaged?
If they are fine, they will proceed to mitosis
What happens in the cell cycle checkpoint - Another checkpoint in mitosis 1
o Are the chromosomes aligned on the spindle?
When can and can’t cells respond to extracellular factors 2
– Cells can only respond to extracellular factors during the G1 phase
– They cannot respond once they have started the S phase
What controls cell cycle progression 1
Cyclin-dependent kinase activity controls cell cycle progression
What do Cyclin-dependent kinase need to be active 1
o To be active, it needs to form a complex with Cyclin.
What does the Cyclin-dependent kinase complex recognise 1
o The complex recognises substrate proteins and is then phosphorylated.
How can you regulate Cyclin-CDK Activity 4
o Cyclical synthesis (gene expression) and destruction (by proteasome). o Post translational modification by phosphorylation – depending on modification site may result in activation, inhibition or destruction o Dephosphorylation o Binding of cyclin-dependent kinase inhibitors
What protein is a key substrate of G1 and G1/S cyclin-dependent kinases 1
The retinoblastoma protein is a key substrate of G1 and G1/S cyclin-dependent kinases
The retinoblastoma protein and S phase 3
Unphosphorylated RB binds E2F, preventing its stimulation of S-phase protein expression.
When phosphorylated by cyclins, RB releases E2F.
The released E2F stimulates expression of more Cyclin E and S-phase proteins e.g. DNA polymerase, thymidine kinase, PCNA etc. DNA replication starts.
The retinoblastoma protein and G0/G1 phase 1
RB in cells that are in g0/g1 adds TF E2F.
What are the 2 families of CKIs (Cyclin-dependent kinase inhibitors) 2
1) CDK Inhibitory Protein/Kinase Inhibitory Protein (CIP/KIP) family (now called CDKN1)
2) Inhibitor of Kinase 4 family (INK4) (now called CDKN2)
CKIs - CDKN1 3
CDK Inhibitory Protein/Kinase Inhibitory Protein (CIP/KIP) family (now called CDKN1)
– Expression of members of this family stimulated weakly by TGF-Beta and strongly by DNA damage (involving TP53)
TP 53 Tumour Suppression gene, activates expression of kinase inhibitors
– Inhibit all other CDK-cyclin complexes (late G1, G2 and M)
– Are gradually sequestered by G1 CDKs thus allowing activation of later CDKs
What does the TP 53 Tumour Suppression gene activate 1
TP 53 Tumour Suppression gene, activates expression of kinase inhibitors
CKIs - CDKN2 3
Inhibitor of Kinase 4 family (INK4) (now called CDKN2)
– Expression stimulated by TGF-Beta
– Specifically inhibit G1 CDKs (e.g. CDK4 the kinase activated by growth factors)
What induces cyclin expression? 1
Growth factors induce cyclin expression
How do growth factors induce cyclin expression - steps 6
- Cells FROM G0 receive GF
- Receptors on cell recognise GF they bind
- Activate signal transducers and in turn intracellular pathways
- Leads to effect in nucleus
- Different waves of transcription factor will be activated
- Regulates expression of P21/Cyclin
Sequential activities in the cycle, triggered by growth factors- steps 8
- Growth factor signalling activates early gene expression (transcription factors – FOS, JUN, MYC)
- Early gene products stimulate delayed gene expression (includes Cyclin D, CDK2/4 and E2F transcription factors)
- E2F sequestered by binding to unphosphorylated retinoblastoma protein (RB)
- G1 cyclin-CDK complexes hypophosphorylate RB and then G1/S cyclin-CDK complexes hyperphosphorylate RB releasing E2F
- G1 CDKs are activated in response to environmental signals, late CDKs by preceding kinase activities.
- E2F stimulates expression of more Cyclin E and S-phase proteins (e.g. DNA polymerase, thymidine kinase, Proliferating Cell Nuclear Antigen etc.)
- S-phase cyclin-CDK and G2/M cyclin-CDK complexes build up in inactive forms. These switches are activated by post-translational modification or removal of inhibitors, driving the cell through S-phase and mitosis.
- Hyperphosphorylated RB is dephosphorylated by protein phosphatase 1. G1 CDKs hypophosphorylate, and late CDKs hyperphosphorylate.
What does DNA damage detected at checkpoints lead to 3
Cell cycle arrest. The cell will stop the cell cycle (with the help of CKIs, CHEK2, etc.).
It will attempt DNA repair (with nucleotides or base excision enzymes, mismatch repair, etc.).
Apoptosis. If repairing is unsuccessful, then the cell is programmed for cell death (via the BCL2 family, capases, etc.).
Do all cells express TP53 1
Tumour suppressor P53, all cells express TP53
What does low or no TP53 mean 1
No DNA damage = low TP53
What does a high expression of TP53 mean 1
High expression = problem with the cell
What happens in response to DNA damage?
o In response to DNA damage, mutagen, kinases atm/atr kinases can detect and then become activates
o Intact DNA molecule has a mutation
o The damage is detected by kinases
o These kinases activate CHEK2 –> TP53 is a substrate for CHEK2
o P53 is expressed in cells however is the protein is not functional as it is quickly degraded by the proteasome.
o In response to DNA damage, kinases phosphorylate P53.
o When it is phosphorylated, it cannot be degraded and it now stabilised and active.
o It will now go and bind the promoters of transcription factors and will help to express genes that are required for DNA repair.
o If a cell cannot be repaired, P53 will trigger apoptosis which gets rids of cells that are deleterious for the whole organism.
• G1 and G1/S Cyclin-CDK complexes phosphorylate RB in the absence of inhibition by CKIs (expression of these is regulated by TP53 or TGF)
• G1 and G1/S Cyclin-CDK complexes phosphorylate RB in the absence of inhibition by CKIs (expression of these is regulated by TP53 or TGF)
