Cellular Growth Regulation Flashcards
What are some considerations for cell growth when looking at the growth of a population of cells?
- Distinguish between increase in cell number (hyperplasia) and cell size (hypertrophy)
- Growth also depends on integration of intracellular and extracellular signals
- (checks on cellular physiology, growth and inhibitory factors, cell adhesion)
What are some considerations for cell growth when looking at the growth at a cellular level?
- Look at the cell cycle
- Cell growth = increase in size and cell division
- Cell cycle phases (G1,S, G2 and M)
- Progression controlled at three key checkpoints (also called arestriction points)
- G1/G2 and M checkpoint
What are some considerations for cell growth when looking at the loss of cells by programmed cell death (apoptosis)?
- A coordinated programme of cell dismantling ending in phagocytosis.
- This is distinct from necrosis!!!
- Occurs during normal development
- E.g. separation of digits, involution, immune and nervous system development
- Occurs in response to DNA damage and infection
Identify growth factors/cytokines/interleukins which stimulate and inhibit proliferation + induce differentiation and induce apoptosis
Stimulate proliferation (also known as mitogens) and maintain survival
- EGF, FGF, Interleukins (IL2 and IL4), NGF (named after orginally identified target)
- PDGF and IGF-1
Stimulate differentiation and inhibit proliferation
TGF- beta
Induce apoptosis
TNF-alpha
What are the three broad classes of interleukins, cytokines and growth factors?
- Paracrine – group of cells produce the growth factor to activate nearby cells locally with the appropriate cell surface receptor
- Autocrine – produced by a cell which also expresses the appropriate cell surface receptor
- Endocrine – growth factors released systemically for distant effects
Describe the phases of the cell cycle
Interphase (G1,S,G2), M phase (Mitosis and Cytokinesis)
- G1 Phase = cell grows larger, copies organelles, accumulates lipids and macromolecules
- S phase = Replication of DNA, duplication of centrosome (helps separate DNA in M phase)
- G2 phase = cell grows further, reorganises its contents in preparation for mitosis
- Mitotic Phase = cell divides forming two daughter cells and undergoes cytokinesis
- Go phase = one of the daughter cells can exit the cell cycle and form quiescent cells, if they receive a mitogen (FGF,EGF), they can re-enter the cell cycle and start dividing or they can remain in the resting state such as neuronal cells these are terminally differentiated.
Recap the process of DNA replication
- DNA is replicated semi conservatively (daughter cells inherit one parental and one new strand)
- New DNA is synthesised in the 5’ to 3’ direction from deoxynucleotide triphosphate precursors at a replication fork by multienzyme complex (a replication machine)
- Fidelity is determined by base pairing (A=T, G=G) and presence of a proof-reading enzyme in DNA polymerase
- Synthesis of new DNA strand uses an RNA primer and occurs continuously on the leading strand and discontinuously on trailing strand (Okazaki fragments which are ligated together after removal of the RNA primer)
Recap the stages of the mitosis
-
Prophase
- Nucleus becomes less definite
- Microtubular spindle apparatus assembles
- Centrioles (yellow) migrate to poles
-
Prometaphase
- Nuclear membrane breaks down
- Kinetochores attach to spindle in nuclear region
-
Metaphase (2)
- Chromosomes (blue) align in equatorial plane
-
Anaphase (3)
- Chromatids separate and migrate to opposite poles
-
Telophase (4)
- Daughter nuclei form
- Cytokinesis
- Division of cytoplasm
- Chromosomes decondense
What is one technique where you can find out the DNA content of a cell?
You can use a fluorescence-activated cell sorter to analyse the cell DNA content.
- Cells are taken and labelled with dye and read by a laser
- Cells that have just started replicating and cells that have finished replicating their DNA
- Number of cells in G2/M phase is greater in a high rate of cell division
What are the two types of drugs that act on the cell cycle?
What are these drugs typically used for?
- S-phase active
- M-phase active
These drugs are typically used for the treatment of cancers - preventing growth of tumours
Give example of S-phase active drugs
- 5-Fluorouracil (analogue of thymidine, blocks thymidylate synthesis)
- Bromodeoxyuridine (another analogue that may be incorporated into DNA and detected by antibodies to identify cells that have passed through the S-phase)
Give examples of M-phase active drugs
- Colchicine = (stabilizes free tubulin, preventing microtubule polymerisation (formation) arresting cells in mitosis – used in karyotype analysis
- Vinca alkaloids
- Palclitaxel (Taxol, stabilizes microtubules, prevents de-polymerization, arrests the cells in mitosis)
Summarise the cell cycle checkpoints
- Progression in the cell cycle is controlled by protein specific kinases and phosphatases ensuring strict alternation of mitosis and DNA replication
- G1 Checkpoint = is DNA damaged, is cell size correct? are there enough metabolites/nutrient stores available
- G2 Checkpoint = Has DNA been replicated correctly without DNA damage
- M phase Checkpoint = Are chromosomes aligned on spindle?
Which stage of the cell cycle is the only stage that is able to respond to mitogens?
The only phase where cells can respond to mitogens is the G1 phase!!
How is the cell cycle controlled?
BY CYCLIN DEPENDANT KINASES
- CDKs will form a complex with kinases which are made active allowing them to phosphorylate target specific substrates
- This will make them more or less active
- Cyclins will activate kinases but will also direct CDKs to a specific set of target proteins appropriate to the cell cycle
- e.g M cyclin direct CDKs to M phase target proteins - to stimulate nuclear membrane breakdown
What is retinoblastoma?
- Key substrate of the G1/S cyclin dependant kinases
How does retinoblastoma play a role in the cell cycle progression?
What molecule does it bind to making it inactive?
- Unphosphorylated RB will bind to E2F making it inactive
- Cyclin D-CDK4 and cyclin E-CDK2 will phosphorylate retinoblastoma and can no longer bind E2F
- E2F is active and binds to promoter regions stimulating further expression of cyclin E and S-phase proteins
- e.g DNA polymerase, thymidine kinase and PCNA
Describe the sequences of events that is triggered by growth factors.
- 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
- 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.
What are the three events that can result from the detection of DNA damage?
- 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.).
- If repairing is unsuccessful, then the cell is programmed for cell death (via the BCL2 family, capases, etc.).
What enzyme is involved in cell DNA damage?
TP53
- DNA damage is detected by kinases which activate CHEK2 (cyclic kinase inhibitor) to prevent continuation of the cell cycle
- Normal TP53 is not present in cells as it is degraded quickly by the proteasome
- Kinases will go on and phosphorylate TP53 causing it to become a stable protein
- This makes it active and expresses genes required for DNA repair
- If TP53 cannot repair the damaged DNA then apoptosis is triggered
