2: The cell cycle Flashcards
1
Q
What factors make cells divide at different rates?
A
- Embryonic vs adult cells (early frog embryo cells - 30 min)
- Complexity of system (yeast cells - 1.5 - 3 h)
- Necessity for renewal
(intestinal epithelial cells - ~20 h
hepatocytes - ~1 year (they are very quiescent but can also strongly unregulated cell division e.g. after lobe removal)) - . State of differentiation (some cells never divide -
i.e. neurons and cardiac myocytes) - Tumour cells?
2
Q
Why is appropriate regulation of cell division imoportant?
A
- Premature, aberrant mitosis results in cell death
- In addition to mutations in oncogenes and tumour suppressor genes, most solid tumours are aneuploid (abnormal chromosome number and content).
- Various cancer cell lines show chromosome instability (loose and gain whole chromosomes during cell division)
- Perturbation of protein levels of cell cycle regulators is found in different tumours - abnormal mitosis (if the proteins that regulate cell division are found in abnormal amounts, this adisregulates cell division)
- Contact inhibition of growth
- Attacking the machinery that regulates chromosome segregation is one of the most successful anti-cancer strategies in clinical use
3
Q
Contact inhibition of growth
A
- cells know when they have to stop dividing.
- In tumours in oncogenesis this is lost, they have no recognition of their neighbours and space and they continue growing.
4
Q
What is the cell cycle? What are the 3 components?
A
Orderly sequence of events in which a cell duplicates its contents
and divides in two.
- duplicaition, division and coordination
5
Q
What happens in the M-phase and in interphase?
A
M: Division
Nuclear division
Cell division (cytokinesis]
I: Duplication
DNA
organelles
protein synthesis
6
Q
When are cells most vulnerable to damage?
A
Mitosis - most vulnerable period of cell cycle:
- Cells are more easily killed (irradiation, heat shock, chemicals)
- DNA damage can not be repaired
- Gene transcription silenced
- Metabolism? (reduced?)
During mitosis gene transcription and metabolism are reduced, the cell is mainly focusing on dividing.
7
Q
What occurs in the S-phase of the cell cycle?
A
- DNA replication
- Protein synthesis: initiation of translation and elongation increased; capacity is also increased
- Replication of organelles (centrosomes, mitochondria, Golgi, etc)
in case of mitochondria, needs to coordinate with replication of mitochondrial DNA
8
Q
What are the phases of the cell cycle?
A
- M: cell division
- G0: cell cycle machinery dismantled (the cell does whatever its function is)
- G1: (gap) decision point -> everything ready and ok for division?
- S: DNA replication, protein and organelle synthesis
- G2: (gap) decision point -> are there any mutations? Are all repairs done?
9
Q
The centrosome
A
- Consists of two centrioles (barrels of nine triplet microtubules)
- Functions: microtubule organizing center (MTOC) and mitotic spindle
- Key organelle and important for cell division.
- Always at 90 degrees to eachother
- Maintained in that position by a cloud around them.
- They will form the microtubule organising centre, coordinate chromosome movement.
10
Q
When are centrosomes duplicated?
A
- centrioles split in G1 phase (usually the two centrioles are at 90 degrees to one another but at that point they separate)
- during S phase they each replicate a second one that is then 90 degrees to itself.
- by G2 they are ready
- they are duplicated because each cell needs to have a centrosome
11
Q
What are the 6 phases of mitosis?
A
- prophase
- prometaaphase
- metaohase
- anaphase
- telophase
- cytokinesis
12
Q
What happens in prophase?
A
- condensation of chromatin, replicated chromosomes condense
- Condensed chromosomes - each consists of 2 sister chromatids, each with a kinetochore
- you start seeing some rods in the nucleus under the microscope
- Duplicated centrosomes migrate to opposite sides of the nucleus and organize the assembly of spindle microtubules
- Mitotic spindle forms outside nucleus between the 2 centrosomes
13
Q
Condensation of chromatin
A
- DNA double helix (2 nm)
- beads on a string form of chromatin (11 nm) -> histone complexes
- 30 nm chromatin fibre of packed nucleosomes
- extended scaffold associated form (300 nm)
- condensed scaffold association form (700 nm)
- chromosome (1400 nm)
=> once duplicated the DNA is packaged to avoid DNA damage/breakage.
14
Q
Centromere
A
- constriction in the middle of the chromosome
- ‘‘belt’’
15
Q
Kinetochore
A
- protein structure
- at the centromere of chromosomes
- microtubules attach to kinetochore to the spindle to pull the chromosome before division
16
Q
Spindle formation
A
- Radial microtubule arrays (ASTERS) form around each centrosome (microtubule organizing centers - MTOC)
- Radial arrays meet
- polar microtubules form
-> microtubules are in a dynamic state.
17
Q
What occurs in metaphase?
A
Chromosomes aligned at equator of the spindle
- line in the Middle (‘‘M’’ like middle and metaphase)
18
Q
What occurs in prometaphase?
A
- early PMP:
- Breakdown of nuclear membrane
- Spindle formation largely complete
- Attachment of chromosomes to spindle via kinetochores (centromere region of chromosome) - late PMP:
- Microtubule from opposite pole is captured by sister kinetochore
- Chromosomes attached to each pole congress to the middle
- Chromosome slides rapidly towards center along microtubules
19
Q
What do the microtubules attach to?
A
- they radiate out from the spindle
- attach to kinetochore on chromosomes
20
Q
CENP-E
A
centromere protein E (kinetochore tension sensing)
-> tension between chromosomes and microtubules
21
Q
What occurs in anaphase?
A
- Paired chromatids separate to form two daughter chromosomes
- Cohesin holds sister chromatids together
- Anaphase A and B
- anaphase A:
- Breakdown cohesin
- Microtubules get shorter
- Daughter chromosomes pulled toward opposite spindle poles
- anaphase B:
1 - Daughter chromosomes migrate towards poles
2 - Spindle poles (centrosomes) migrate apart
22
Q
What are the 2 movements in anaphase B? Why are they important?
A
1: the chromosomes are being pulled towards the poles
2: the spindle poles (centrosomes) migrate apart in opposite directions.
=> important because you don’t want anything there while the cell is dividing because the constriction occurs in the middle so these mechanisms are important to allow for the chromosomes to go where they should.
23
Q
What happens in telophase?
A
- Daughter chromosomes arrive at spindle
- Nuclear envelope reassembles at each pole
- Assembly of contractile ring (of actin and myosin filaments)
24
Q
What is the contractile ring in telophase made of?
A
actin and myosin filaments
25
What occurs in cytokinesis?
- the cell divides -> 2 independent cells
- there is a midbody in the centre (kind of like tails of the cells)
- chromosomes begin to decondense and nuclear structures reform
26
Transition out of metaphase: Spindle Assembly checkpoint
- unattached kinetochores generate signals (normal in prometaphase, should not be the case in metaphase)
- requires: CENP-E; BUB protein kinases
- BUBs dissociate from kinetochore when chromosomes are properly attached to the spindle
- When all dissociated, anaphase proceeds.
- there is a kinase attached to the kinetochore -> when there is no attached microtubule it sends signals
Important checkpoint in terms of aneuploidy
27
What are the possible (mis-)attachments of microtubules of kinetochores?
- amphelic (normal) attachment: a microtubule attaches to a kinetochore and a microtubule from the opposing pole attaches to the kinetochore of the sister chromatid (does not produce signal)
- syntelic attachment: 2 microtubules from one pole attach to the kinetochores of the same chromosome (both sister chromatids) this will make both go to the same daughter cell and the other one will lack this chromosome -> may or may not produce a checkpoint signal;
- monotonic attachment: only one of the sister chromatids kinetochore has an attachment with a microtubule (this will produce a checkpoint signal)
- merotelic attachment: one sister chromatid has an attachment to one pole and the other sister chromatid has attachments to 2 opposing poles. -> this does not produce a signal; can cause breakage of the chromosome or chromosome loss at cytokinesis)
28
What can cause aneupliody?
(a) mis-attachment of microtubules to kinetochores
| (b) aberrant centrosome/DNA duplication
29
aberrant centrosome/DNA duplication
- e.g. 4 centrosomes formed
- uneven distribution of chromosomes accross 4 instead of 2 cells
- 4 centrosomes, this messes up the spindles, chromosomes don’t know where to go.
- aberrant cytokinesis
30
Anti-cancer therapy by inducing gross chromosome mis-segregations
Checkpoint kinase (CHKE1 and CHKE2) – Serine threonine kinase
- activation holds cells in G2 phase until all is ready
- inhibition leads to untimely cell transition to mitosis
Taxanes and vinca alkaloids (breast and ovarian cancers)
- Alters microtubule dynamics
- Produces unattached kinetochores
- Causes long-term mitotic arrest.
=> induce the cells of faking that they are ready to divide, increases the chance of an inviable cell with a high chance of cell death. (however affects all rapidly dividing cells of the body (but because cancer cells divide so rapidly they will be more affected proportionately)
31
What happens if something goes wrong in the cell cycle?
e.g. Cell is not big enough or DNA damage
1. Cell cycle arrest
at check points (G1 and spindle check point)
can be temporary (i.e. following DNA repair)
2. Programmed Cell Death / Apoptosis
DNA damage too great and cannot be repaired
Chromosomal abnormalities
Toxic agents
Cell cycle progression aborted and cell destroyed
32
What are CHKE1 and CHKE2 responsible for?
- holding the cell in G2 until it is ready to divide
- they are tyrosine kinases
- if they are inhibited (as a part of cancer therapy) they will allow the cell to go into mitosis and the cell will apoptosis because of the gross misalignment and not being ready.
33
Taxanes and vinca alkaloids
- (breast and ovarian cancers) in particular
- Alters microtubule dynamics, interfere with/block spindle formation
- Produces unattached kinetochores -> lots of signals
- Causes long-term mitotic arrest, the cells die because they are vulnerable at that time.
34
What are the cell cycle checkpoints?
- Metaphase checkpoint (exit from M I): checks for sister chromatid alignment at the equatorial plate. Is it okay for mitosis to occur?
- G1 checkpoint: mainly driven by growth factors, say if everything is okay to proceed
- G2 checkpoint: entry to mitosis. Is there any DNA damage? Can we proceed to mitosis?
35
How do tumours interfere with cell cycle checkpoints?
- G1 CP: they can upregulate proteins, receptors, signals and increase the frequency and speed of cell cycles
- G2 CP: the cells enter mitosis with DNA that is not ready to go
- Metaphase CP: block alignment which can mean that you lose or gain chromosomes.
36
De-regulation of cell cycle during tumorigenesis
- after cell division cells often go into the G0 phase where they do their business and fulfil their function and dismantle their apparatus
- tumour cells can avoid this and make the cells just keep dividing and not go into the G0 phase.
37
G0 phase and also: what triggers the cell to enter the cell cycle and divide?
- In the absence of stimulus, cells go into Go (quiescent phase)
- Most cells in the body which are differentiated to perform specific functions
- Cells are not dormant, but are non-dividing
- Exit from G0 highly regulated - requires growth factors and intracellular signalling cascades
- Overproliferation in cancer.
- Growth factors can regulate the entry into the growth cycle.
38
Signalling through the cell to initiate entry into the cell cycle
Signalling cascades:
- Response to extracellular factors (e.g. growth factors)
- Signal amplification
- Signal integration
- Modulation by other pathways
- Regulation of divergent responses (e.g. regulation of gene expression, regulation of metabolic pathways, changes in cytoskeleton)
39
Signalling by peptide growth factors
- Ligand binds and activates the receptor
- Epidermal growth factor (EGF); Platelet-derived growth factor (PDGF)
- Respective receptors found as monomeric, inactive state
- Receptor Protein Tyrosine Kinase (RPTK)
- In the presence of a ligand the receptors form dimers (cross-phopshorylation?) and are activated by phosphorylation (Phosphorylated amino acid residues in kinase domain)
- receptor activation triggers: kinase cascades
binding of adapter proteins
40
Protein phosphorylation - what amino acids can it occur at?
- need an exposed OH group -> this is removed and via ATP and a kinase phosphorylated
- Serine, Threonine, Tyrosine
- The added phosphate group (negatively charged) can alter protein function by:
a) causing a change in shape (conformation) leading to change in activity (+ve or –ve)
b) creating a docking site for another protein
41
What reverses what a kinase did?
- a phosphatae
| - it removes the phosphate (Pi) and there is an OH group instead
42
Signalling by peptide growth factors: receptor activation triggers different phosphorylation events
- receptor activation triggers different phosphorylation events
- in the presence of a ligand: Receptors form dimers
are activated by phosphorylation
- receptor activation triggers: kinase cascades
binding of adapter proteins -> there are also other phosphorylation sites.
- Frequently the protein regulated by a kinase is another kinase, and so on… (protein kinase cascade)
- Leads to signal amplification, diversification and opportunity for regulation
- Flash of activation and then it comes back to the normal situation by phosphatase (reversal function of a kinase)
Returns it to the normal kinase.
