Cell growth and differentiation Flashcards
What are growth factors, cytokines and interleukins?
What are their functions?
Proteins which:
- stimulate proliferation (called mitogens) and maintain survival
- stimulate differentiation and inhibit proliferation (e.g. TGFβ)
- induce apoptosis (e.g. TNF⍺ and other members of the TNF family)
Where are growth factors, cytokines and interleukins derived from?
From 3 different sources:
- Paracrine: produced locally to stimulate proliferation of a different (nearby) cell type with 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 effects
Phases of the cell cycle
Mitotic (M) phase
Interphase (G1,S,G2)
What happens in the M phase of the cell cycle?
- Separation of chromosomes into two sets
- Cell divides its cytoplasm, forming two new cells
What happens during interphase?
G1:
-cells grow physically larger and copies organelles
S phase:
-cell synthesises a complete copy of DNA in its nucleus and duplicates the centrosome, which helps separate the DNA during M phase
G2 phase:
-cell grows more, makes proteins and organelles, and begins to reorganise its contents in preparation for mitosis
What is the G0 phase?
After cell division, some cells may exit the G1 phase and enter a resting state (G0 phase). Here, cells are not actively preparing to divide and are considered quiescent.
What is the fate of G0 cells?
- Re-enter G1 phase in presence of mitogens
- Terminal differentiation (post-mitotic cells) which eventually undergo cell shedding & apoptosis
How is the rate of cell division measured?
Fluorescence-activated cell sorting (FACS) is used to determine the rate of cell division and the phase of the cell cycle at which cells are at.
Fluorescence activated cell sorting: Steps
1) Cells are obtained
2) DNA is labelled with fluorescent dye
3) Fluorescent dye is read by a laser
4) Laser provides information about DNA content by analysing how fluorescent the nuclei are
The DNA content is greater is G2/M phase because of the replication at S phase
Stages of mitosis
Four main stages:
1) Prophase/Prometaphase
2) Metaphase
3) Anaphase
4) Telophase
Prophase
- nucleus becomes less definite
- microtubular spindle apparatus assembles
- centrioles migrate to poles
Prometaphase
- nuclear envelope breaks down
- kinetochores attach to spindle via microtubules
Metaphase
chromosomes align in equatorial plane
Anaphase
chromatids separate and migrate to opposite poles
Telophase
daughter nuclei form
What is after mitosis?
cytokinesis
Cytokinesis
- division of the cytoplasm to form two separate daughter cells
- chromosomes decondense
5-Fluorouracil
S-Phase Active Drug:
-thymidine analogue which blocks thymidylate synthesis, preventing thymidine production and therefore preventing DNA replication
*used in cancer treatment
Bromodeoxyuridine (BrdU)
S-Phase Active Drug:
-thymidine analogue that is incorporated into DNA and detected by antibodies to identify proliferating cells that have passed through the S-phase
Colchicine
M-Phase Active Drug:
- stabilises free tubulin, preventing microtubule polymerisation and arresting cells in mitosis
- used in karyotype analysis
Vinca alkaloids
M-Phase Active Drug:
-like colchicine, stabilises free tubulin, preventing microtubule polymerisation and arresting cells in mitosis
*used in cancer treatment
Paclitaxel (Taxol)
M-Phase Active Drug:
-stabilises microtubules, preventing de-polymerisation and cells will be arrested in the M-phase
*used in cancer treatment
Cell cycle checkpoints
There are controls (involving specific protein kinases and phosphatases) that ensure the strict alternation of mitosis and DNA replication
G1,G2,M checkpoint
*learn diagram
What controls cell cycle progression?
Cyclin-dependent kinase activity
Cell cycle regulators
· Cyclin Dependant Kinases (CDKs)- enzymes that phosphorylate the target proteins. They become active when bound to a corresponding cyclin.
· Cyclins- regulators of CDKs. Different cyclins are produced at each phase of the cell cycle.
Regulation of Cyclin-CDK Activity
· Cyclin synthesis (gene expression) and destruction (by proteasome)
· Post-translational modification by phosphorylation- depending on the modification site may result in activation, inhibition or destruction
· Dephosphorylation
· Binding of cyclin-dependent kinase inhibitors
The function of retinoblastoma protein in cell cycle
pRB is unphosphorylated and binds E2F transcription factor preventing its stimulation of S-phase protein expression
>inhibits S-phase
What affects the binding of pRB to E2F?
In presence of Cyclin D-CDK4 complex or Cyclin E-CDK2 complex, retinoblastoma protein becomes phosphorylated,
it is no longer able to bind E2F. E2F is therefore released and stimulates the expression of more Cyclin E and S-phase proteins e.g. DNA polymerase, thymidine kinase, Proliferating Cell Nuclear Antigen etc.
>DNA replication then starts.
Cyclin Dependent Kinase Inhibitors (CKIs)
Two families:
CDK Inhibitory Protein/Kinase Inhibitor Protein (CIP/KIP) Family
>now called CDKN1
Inhibitor of Kinase 4 Family (INK4)
>now called CDKN2
CDK Inhibitory Protein/Kinase Inhibitor Protein (CIP/KIP) Family
- 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
Inhibitor of Kinase 4 Family (INK4)
- Expression stimulated by TGFβ
- Specifically inhibit G1 CDKs (e.g. CDK4 the kinase activated by growth factors)
What induces cyclin expression?
Growth factors (mitogens)
How do growth factors (mitogens) induce cyclin expression?
1) Growth factor binds growth factor receptor on cell membrane
2) Signal transducers are activated- which are a cascade of different components which eventually affect waves of transcription factor activation/expression in the nucleus
3) mRNA produced which then codes for a protein with specific effects
Effect of growth factor signalling on gene expression
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).
What hypophosphorylates pRB?
G1 cyclin-CDK complexes hypophosphorylate pRB
What hyperphosphorylates pRb?
G1/S cyclin-CDK complexes hyperphosphorylate pRB releasing E2F
How is hyperphosphorylated pRB dephosphorylated?
by protein phosphatase 1
Which cyclin-CDK complexes build up in inactive forms?
S phase and G2/M phase cyclin-CDK complexes
-switches are activated by post-translational modification or removal of inhibitors, driving the cell through S-phase and mitosis
Which CDKs are activated in response to environmental signals?
G1 CDKs
Function of different CDKs
G1 CDKs hypophosphorylate
Late CDKs hyperphosphorylate
Effect of DNA damage in cell cycle
DNA damage detected at checkpoints triggers cell cycle arrest or apoptosis
Essential transcription factor in response to DNA damage is…
TP53
TP53 in response to DNA damage
1) Mutagen causes DNA damage which activates kinases, and in turn activates CHEK2
2) This causes phosphorylation of TP53
3) TP53 phosphorylation allows it to survive and it is no longer quickly degraded, becoming a stable protein
4) TP53 is now active and binds promoters of transcription factors to express genes required for DNA repair
5) If DNA cannot be repaired, TP53 triggers apoptosis
6) TP53 also expresses CKIs to induce cell cycle arrest
