Cellular growth regulation Flashcards
1
Q
What are GFs, cytokines and ILs?
A
- Proteins that stimulate proliferation and maintain survival (called mitogens)
- Named after target tissue (FGF, EGF)
- Or named after origin (platelet-derived GF)
- Stimulate differentiation and inhibit proliferation eg TGFb
- Induce apoptosis eg TNFa
2
Q
What are the three broad classes of mitogens?
A
- 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 (can stimulate its own growth - +ve feedback)
- Endocrine = like conventional hormones, released systemically for distant effects
3
Q
How can you affect the cell population growth with GFs?
A
- Adding a GF to a set of dormant cells will cause an increase in growth of cells
- If you remove the GF, they’ll stop – need continuous GF presence to continue the growth of cells
- Adding a GF inhibitor such as TGFβ, then the growth will stop
- Finally you can reduce the cell number again by adding a death signal, such as TNFα
4
Q
What are the phases of the cell cycle?
A
- G1 = Gap phase 1, cell synthesises mRNA and proteins ready for subsequent stages ploidy=2N
- S phase = DNA replicated
- G2 = cell continues to grow and synthesise new proteins - ploidy=4N
- Mitosis = Cell division
- G1/G0 - G0 = cells go into quiescent state. Some will then terminally differentiate to become post-mitotic cells and then will die by apoptosis
5
Q
What stages can we distinguish under the microscope?
A
- Mitosis - when sister chromatids are being pulled apart by spindles - can see the chromosomes as they condense
- Can incorporate radioactive thymidine into actively synthesising DNA during S phase
- Can see the different amounts of DNA present (pre-/post-mitosis) using FACS
6
Q
How do we use FACS to analyse DNA content?
A
- Incorporate a dye that intercalates between the bases and the DNA
- Cells in G1 will have a certain level of fluorescence and cells in G2/M will have twice as much
- The cells in S phase will have varying levels of fluorescence depending on the rate of cell division (more DNA in S if high rate) - can distinguish between low and high rate
7
Q
What is the process of DNA replication?
A
- DNA is replicated semiconservatively (daughter cells inherit one parental and one new strand)
- 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)
- Fidelity is determined by base pairing (A=T, G≡C) and presence of a proof reading enzyme in DNA polymerase
- 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 using DNA ligase)
8
Q
What are the stages of mitosis?
A
• Prophase (1) – Nucleus becomes less definite – Microtubular spindle apparatus assembles – Centrioles migrate to poles • Prometaphase – Nuclear membrane breaks down – Kinetochores attach to spindle in nuclear region • Metaphase (2) – Chromosomes align in equatorial plane • Anaphase (3) – Chromatids separate and migrate to opposite poles • Telophase (4) – Daughter nuclei form • Cytokinesis – Division of cytoplasm – ring of actin between two cells – Chromosomes decondense
9
Q
What drugs act on the S-phase?
A
5-fluorouracil = thymidine analogue
- 5-fu has similar structure to thymidine, but Fl instead of CH3, causing inhibition
Bromodeoxyuridine = another analogue that can be incorporated into the DNA and detected by abs to identify cells that have passed through S-phase
Tamoxifen = oestrogen analogue that blocks its stimulatory effect on growth, inhibiting entry into S-phase
10
Q
What drugs act on m-phase?
A
- Colchine = stabilises free tubulin preventing microtubule polymerisation and arresting cells in mitosis - used in karyotype analysis
- Vinca alkaloids - similar to colchine
- Paclitaxel - stabilises microtubules preventinge depolymerisation
11
Q
Which of the previous drugs are often used in cancer treatment?
A
- 5-fluorouracil, paclitaxel, vinca alkaloids and tamoxifen
- If you can block cells entering the S phase, or inhibit mitosis, you can stop cancerous growth
12
Q
What are the cell cycle checkpoints?
A
- Controls, involving specific kinases and phosphatases, ensure the strict alternation of mitosis and DNA replication
- Series of restriction points where the cell may be arrested if the growth isnt appropriate
- G1/S - check for DNA damage, appropriate growth and adequate metabolite/ nutrient stores
- G2/M - checks for DNA damage and that it has completely replicated
- M - check chromosomes are aligned on spindle
13
Q
What is involved in the G1 checkpoint?
A
- Unphosphorylated Rb binds to E2F, inhibiting it
- E2F is a TF that stimulates entry to S-phase
- CDK4-CyclinD phosphorylates Rb, causing it to be released from E2F
- E2F stimulates entry to the S phase by activating CDK2-Cyclin E, increasing expression of DNA polymerase, thimidine kinase etc
14
Q
What are the 2 families of Cyclin-dependent kinase inhibitors?
A
CDKN1 (CDK inhibitory protein)
- expression of members of this family are stimulated weakly by TGFbeta and strongly by DNA damage
- inhibit all other CDK-cyclin complexes (late G1, G2 and M)
- Are gradually sequestered by G1 CDKs, thus allowing activation of later CDKs
CDKN2 (inhibitor of kinase 4 family)
- Expression stimulated by TGFbeta
- Specifically inhibit G1 CDKs (CDK4)
15
Q
How do GFs induce cyclin expression?
A
- Triggering of cell to enter cycle by exposure to a GF
- GF binds to receptor
- Receptor has signal transduction which causes a serine kinase cascade (MAP kinases), which can cause the expression of a set of genes – waves of gene expression
- The proteins made are transcription factors, which can then move back and synthesise other proteins
16
Q
What is the sequence of events triggered by GFs?
A
- GF signalling activates early gene expression (transcription factors – FOS, JUN, MYC - oncogenes)
- Early gene products stimulate delayed gene expression (includes Cyclin D, CDK2/4 and E2F transcription factors)
- E2F sequestered by binding to unphosphorylated RB protein
- 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.)
17
Q
What do we do if we detect DNA damage?
A
- At each stage you can have checks to ensure that the cell is in an appropriate state to progress – check for DNA damage
- If some is detected, or at the end of G1 the cell hasn’t made enough nutrients/proteins, then the CDK inhibitors will be expressed, causing the arrest of the cell cyle
- If the DNA has been damaged, the cell attempts DNA repair – different mechanisms depending of type of damage (nucleotide of base excision enzymes or mismatch repair etc)
- If the DNA damage is too extensive, then apoptosis can be induced
18
Q
What is TP53?
A
- TP53 (P53) is synthesized by rapidly degraded in the proteasome in most normal cells
- However in a cell that has been damaged, kinases will phosphorylate TP53, stablising it and activating it as a TF.
- This causes expression of CDKI, arresting the cell cycle
- Also activates mechanisms to try and repair DNA
- Or if repair isn’t possible, it will activate the BCL-2 mechanism and cause apoptosis
19
Q
Summary of key steps
A
- Growth factors binding to receptors induce gene expression
- 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)
- E2F released, stimulating expression of genes required for S-phase
- Cell replicates DNA (expression of S-phase Cyclin-CDK complexes)
- If all DNA replicated, G2/M Cyclin-CDK complexes cause cell to enter mitosis. If chromosomes aligned on spindle, exit from mitosis is triggered
- If process fails and repair is not possible, TP53 initiates apoptosis
