Cellular Growth Regulation Flashcards
What are the general considerations for cell growth?
- Growth of a cell population
- Growth at cellular level (cell cycle)
- Loss of cells via apoptosis
What are the 2 ways a cell population can grow?
Distinguish between:
- increase in cell numbers (hyperplasia)
- increase in cell size (hypertrophy)
What is the growth of a cell population dependent upon?
Depends on integration of intra- and extracellular signals (checks on cellular physiology, growth and inhibitory factors, cell adhesion etc.)
What is cell growth?
Cell growth = increase in size (sometimes growth refers to this only) and cell division
What are the phases of the cell cycle?
Cell cycle phases (G1, S, G2, and M)
How is the cell cycle mediated?
Progression controlled at three key checkpoints (restriction points)
What is apoptosis?
A coordinated program of cell dismantling ending in phagocytosis
Distinct from necrosis
When does apoptosis occur?
Occurs during normal development (e.g. separation of the digits, involution, immune and nervous system development)
And in response to DNA damage and viral infectio
Outline the role of growth factors, cytokines and interleukins
These are proteins that:
- Stimulate proliferation (called mitogens) and maintain
survival - Stimulate differentiation and inhibit proliferation e.g.
TGFβ - Induce apoptosis e.g. TNFα and other members of the
TNF family
How are proliferation stimulating proteins named?
Usually named after originally identified target
e.g.
EGF, FGF, Interleukins (IL2 & IL4), NGF
but see also:
PDGF (platelet-derived GF) IGF1 (Insulin-like GF – the main effector of pituitary growth hormone)
What are the broad 3 classes of growth factors, interleukins and cytokines?
- paracrine
- autocrine
- endocrine
What is meant by paracrine?
Paracrine: produced locally to stimulate proliferation of a different cell type that has the appropriate cell surface receptor
What are endocrine signlas?
Endocrine: like conventional hormones, released systemically for distant effects
What is meant by autocrine signalling?
Autocrine: produced by a cell that also expresses the appropriate cell surface receptor
What is the effect of PDGF on the cell cycle?
PDGF - platelet derived growth factor
PDGF presence: Cells start entering cell cycle and proliferating
PDGF no longer available: cells stop dividing = plateau
What is the role of TGFβ in the cell cycle?
TGFβ - transforming growth factor beta
Causes proliferation and induction into cell cycle
How does TNF𝛼 effect the cell cycle?
TNF𝛼 - Tumor necrosis Factor
Eventually cells receive death signal and enter apoptosis
Cell no. decreases
Outline what occurs during interphase of the cell cycle
Cells grow in size as most macromolecules are synthesized continuously throughout interphase
Occurs after mitosis
When do cells enter G0 phase?
When cells don’t receive FGF they become quiescent cells
How do quiescent cells re-enter the cell cycle?
Re enter cell cycle when exposed to growth factors
How can we identify the no. of cells present during the cell cycle?
Use Fluorescence Activated Cell Sorter Analysis of Cell DNA Content
How does Fluorescence Activated Cell Sorter Analysis of Cell DNA Content work?
Cell DNA labelled with a fluorescent dye
The dye is read by a laser which tells us the DNA content present ∴ cell no.
What result would fluorescence show for cells that grow slow
Cells that grow slowly will show a higher G1 peak as most cells are still in that phase
For fast growing cells, what would the fluorescence results show?
Fast cell division will have less cells in the G1 phase as the cells are progressing through the cell cycle
Outline the stages of DNA replication
- DNA replicated semiconservatively
(daughter cells inherit one parental and one new strand) - New DNA synthesized in 5’-3’ direction from
deoxynucleotide triphosphate precursors at a replication
fork by a multienzyme complex (a replication machine) - Fidelity is determined by base pairing (A=T, G≡C) and
presence of a proof reading enzyme in DNA polymerase - Synthesis of new DNA strand uses an RNA primer and
occurs continuously on leading strand and
discontinuously on trailing strand
(giving rise to Okazaki fragments, which are ligated
together after removal of the RNA primer)
What are the main stages of Mitosis?
1. Prophase
prometaphase
2. Metaphase
3. Anaphase
4. Telophase
Cytokinesis
Explain what happens during prophase
Nucleus becomes less definite
Microtubular spindle apparatus assembles
Centrioles migrate to poles
What happens in prometaphase?
The nuclear membrane breaks down
Kinetochores attach to spindle in nuclear region
Describe the events of Metaphase?
Chromosomes align in the equatorial plane
Explain what occurs during anaphase
Chromatids separate and migrate to opposite poles
What happens during telophase?
Daughter nuclei form
What is cytokinesis?
Division of cytoplasm
chromosomes decondense
Name drugs that act on the S-phase of the cell cycle
- 5-Fluorouracil
- Bromodeoxyuridine (BRDU)
Explain the role of 5-Fluorouracil on the S-phase of the cell cycle
5-Fluorouracil is an analogue of thymidine and blocks thymidylate synthesis
Why is BRDU used to act on the S-phase of the cell cycle?
Bromodeoxyuridine (BRDU) is also an analogue that may be incorporated into DNA
Can be detected by antibodies to identify cells that have passed through the S-phase
- ab recognise BRDU and can be added to samples to identify the DNA being produced
Which drugs act on the M-phase of the cell cycle?
- Colchicine
- Vinca alkaloids
- Paclitaxel
What is the effect of Colchine on M-phase?
stabilizes free tubulin, preventing microtubule polymerization and arresting cells in mitosis
When is Colchine used to act on the M-phase?
Colchine used in karyotype analysis
What is the role of Vinca alkaloids?
Similar action to colchine
What is the effect of Paclitaxel on M-phase?
Taxol, stabilizes microtubules, preventing de-polymerization
What treatment uses drugs that act on the M-phase of the cell cycle?
5-Fluorouracil, paclitaxel, the vinca alkaloids and tamoxifen are used in treatment of cancer
What are cell cycle checkpoints?
Controls (involving specific protein kinases and phosphatases)
What is the role of cell cycle checkpoints?
They ensure the strict alternation of mitosis and DNA replication
What are protein kinases?
proteins that phosphorylate proteins to activate them
What are phosphatases?
Enzymes that removes phosphate groups from proteins
What are mitogens?
A small protein, that induces a cell to begin cell division
What other regulations are in place to ensure correct cell cycle functioning?
Cells only respond to the extracellular environment during G1 phase - can respond to mitogens
Once they enter S phase cells are unresponsive to extracellular signals
What is the purpose of cyclin-dependent kinase activity?
controls cell cycle progression by phosphorylating specific substrates
How does a cyclin-dependent kinase activity regulate the cell cycle?
CDK-Cyclin complex must form to activate the CDK unit which produces a substrate protein and phosphorylation
How is cyclin-CDK activity itself regulated?
Cyclical synthesis (gene expression) and destruction (by proteasome)
Post translational modification by phosphorylation
– depending on modification site may result in activation, inhibition or destruction
Dephosphorylation
Binding of cyclin-dependent kinase inhibitors
What is the retinoblastoma Protein?
The retinoblastoma protein is a key substrate of G1 and G1/S cyclin-dependent kinases
Explain the role of retinoblastoma protein on the cell cycle
- Unphosphorylated RB binds E2F preventing its
stimulation of S-phase protein expression - Released E2F stimulates expression of more Cyclin E
and S-phase proteins e.g. DNA polymerase, thymidine
kinase, PCNA etc. - DNA replication starts.
What are the 2 families of CDK inhibitors?
- CDK Inhibitory Protein/Kinase Inhibitory Protein (CIP/KIP)
family
(now called CDKN1) - Inhibitor of Kinase 4 family (INK4) (now called CDKN2)
How are CDKN1 (CIP/KIP) expressed?
Expression of members of this family stimulated weakly by TGFβ and strongly by DNA damage (involving TP53)
Inhibit all other CDK-cyclin complexes (late G1, G2 and M)
Are gradually sequestered by G1 CDKs thus allowing activation of later CDKs
How do CIP/KIP CDKN1 inhibit CDKs?
Inhibit all other CDK-cyclin complexes (late G1, G2 and M)
Are gradually sequestered by G1 CDKs thus allowing activation of later CDKs
What regulates teh expression of CDKN2 or INK4s?
Expression stimulated by TGFβ
What is the role of INK4s / CDKN2?
Specifically inhibit G1 CDKs (e.g. CDK4 the kinase activated by growth factors)
Outline how growth factors induce cyclin expression
- Cells enter the cell cycle and receive growth factors in
response to mitogen - The growth factor will bind to the growth factor receptor
on the cell membrane to activate signal transducers - The signal transducers activates a cascade of reactions
resulting in the formation of a perfect nucleus - The nucleus undergoes waves of transcription factor
activation to express RNA to encode genes and
proteins
How are CDKs activated for expression?
G1 CDKs are activated in response to environmental signals, late CDKs by preceding kinase activities.
How does RB phosphorylation regulate cell cycle?
G1 CDKs hypophosphorylate RB
Late G1/S CDKs hyperphosphorylate RB releasing E2F
Hyperphosphorylated RB is dephosphorylated by protein phosphatase 1
What is the result of growth factor signalling?
Growth factor signalling activates early gene expression (transcription factors – FOS, JUN, MYC)
What is activated by the early gene products induced by growth factors?
Early gene products stimulate delayed gene expression (includes Cyclin D, CDK2/4 and E2F transcription factors)
How is E2F sequestered?
E2F sequestered by binding to unphosphorylated retinoblastoma protein (RB)
How does E2F release fluctuate during the cell cycle?
G1 cyclin-CDK complexes hypophosphorylate RB and then G1/S cyclin-CDK complexes hyperphosphorylate RB releasing E2F
What is the role of E2F?
E2F stimulates expression of more Cyclin E and S-phase proteins (e.g. DNA polymerase, thymidine kinase, Proliferating Cell Nuclear Antigen etc.)
How are S-phase and G2/M phase cyclin-CDK complexes readily available?
S-phase cyclin-CDK and G2/M cyclin-CDK complexes build up in inactive forms
How are S/G2/M phase Cyclin-CDK complexes activated?
These switches are activated by post-translational modification or removal of inhibitors, driving the cell through S-phase and mitosis.
When is DNA damage in the cell cycle detected?
DNA damage detected at pre S-phase
Pre M-phase and G2 doublechecks for DNA Damage
Outline the result of DNA damage detection at a checkpoint
DNA damage detected at checkpoints triggers cell cycle arrest or apoptosis as cells will inhibit cyclin because DNA damage will activate protein kinases
How does DNA damage occur?
DNA strand is introduced to a mutagen causing a strand break or base pair mismatch
How is DNA damage detected at the checkpoints?
Mutation is detected by kinases (ATM, ATR) which activates CHEK2 and P53 TF
What is the normal outcome of P53 TF?
P53 TF causes expression of TP53 in cells but it is normally non-functional as it is quickly degraded by the proteasome
How is P53 TF activity altered due to mutation and DNA damage?
DNA damage causes kinase (CHEK2) activation, which phosphorylates TP53 inhibiting its degradation
What is the consequence of activated TP53?
Activated TP53 will bind to TF promoters required for DNA repair
If the DNA damage cannot be prepared, P53 triggers apoptosis to remove mutated cells
What enables gene expression to occur?
Growth factors binding to receptors induce gene expression
How is RB phosphorylated?
G1 and G1/S Cyclin-CDK complexes phosphorylate RB in the absence of inhibition by CKIs (expression of these is regulated by TP53 or TGFβ)
What does E2F release cause?
E2F released, stimulating expression of genes required for S-phase
What enables DNA replication to occur during the cell cycle?
Cell replicates DNA (expression of S-phase Cyclin-CDK complexes)
What are the requirements for a cell to enter mitosis?
If all DNA replicated, G2/M Cyclin-CDK complexes cause cell to enter mitosis
When do cells exit mitosis?
If chromosomes aligned on spindle, exit from mitosis is triggered
What is the consequence of failed cell cycle?
If process fails, TP53 initiates apoptosis
