10. Cellular Growth Regulation Flashcards
What are general considerations for cell growth?
• Growth of a population of cells
- 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.)
• Growth at the cellular level (the cell cycle)
- Cell growth = increase in size (sometimes growth refers to this only) and cell division
- Cell cycle phases (G1, S, G2, and M)
- Progression controlled at three key checkpoints (restriction points)
• Loss of cells by programmed cell death (apoptosis)
- 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 are growth factors, cytokinds and interleukins? and what are their classes?
• PROTEINS THAT:
- stimulate proliferation (called mitogens) and maintain survival
- 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)
- stimulate differentiation and inhibit proliferation e.g. TGF-beta
- induce apoptosis e.g. TNFα and other members of the TNF family
• 3 BROAD CLASSES:
- 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 effects
Cell population growth in response to hormones.
- The cells will respond to PDGF and they will enter the cell cycle. (They will leave the g0 phase)
- The growth inhibitor TGF-beta will inhibit growth
- Apoptosis signal TNF-alpha will decrease the cell number
What happens after mitosis?
- Some of the cells will arrest in the G0 phase
- Some of the cells can also be differentiated.
Describe the difference in DNA content in cell when rate of cell division is low compared to high
- DNA content in G1 phase will decrease in high rate of cell division
- DNA content in S phase will increase in high rate of cell division
- DNA content in G2/M STAYS THE SAME in both high and low rate of cell division
Describe the events of DNA replication
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)
Describe the 4 main stages of mitosis.
• PROPHASE (1) - 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
Expand on the drugs acting on the cell cycle
• S-PHASE ACTIVE
- 5-Fluorouracil (an 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).
• M-PHASE ACTIVE
- Colchicine (stabilizes free tubulin, preventing microtubule polymerization and arresting cells in mitosis – used in karyotype analysis)
- Vinca alkaloids (similar action to colchicine)
- Paclitaxel (Taxol, stabilizes microtubules, preventing de-polymerization)
-5-Fluorouracil, paclitaxel, the vinca alkaloids and tamoxifen (antagonist of oestrogen) are used in treatment of cancer
What are cell cycle checkpoints? And when are they found?
• Controls (involving specific protein kinases and phosphatases) ensure the strict alternation of mitosis and DNA replication.
• Found:
- During most of G1 phase - cells are responsive to growth factors ~ main site of control for cell growth. (cells only respond to mitogens in the G1 phase).
- At the end of G1 phase. Restriction point: DNA not damaged, cell size, metabolite/nutrient stores
- B/w G2 and M phase: DNA completely replicated, DNA not damaged.
- M phase: Chromosomes aligned on spindle
How does cyclin-dependent kinase activity control cell cycle progression?
• There is a:
- CDK catalytic subunit (10 genes)
- Cyclin regulatory subunit (more than 20 genes)
• Active cyclin-CDK complex phosphorylates specific substrates
• Regulation of cyclin-CDK activity:
- 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
Why is the retinoblastoma protein important?
• It is a key substrate of G1 and G1/S cyclin-dependent kinases
- Unphosphorylated RB binds E2F preventing its stimulation of S-phase protein expression
- In the presence of Cyclin D-CDK4 and Cyclin E-CDK2…
- 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 cyclin-dependent kinase inhibitors?
• Two families of CKIs:
1) 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)
- Inhibit all other CDK-cyclin complexes (late G1, G2 and M)
- Are gradually sequestered by G1 CDKs thus allowing activation of later CDKs
2) 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)
How do growth factors induce cyclin expression?
- Growth factor binds to its receptor
- This releases an intracellular signal.
- This causes a kinase cascade
- Causes waves of transcription factor activation in the nucleus
- mRNA is produced and exported out of the nucleus
- Proteins are then produced.
What is the sequence of events 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 does DNA damaged detected at checkpoints trigger in the cell cycle?
- Stop the cycle (cyclin dependent kinase inhibitors, CHEK2 etc.)
- Attempt DNA repair (nucleotide or base excision enzymes, mismatch repair etc.)
- Programmed Cell Death if repair impossible (BCL2 family, caspases)
