Cancer 4: The cell cycle and its regulation Flashcards
What might determine rate of differentiation
Embryonic vs adult cells (early frog embryo cells - 30 min)
Complexity of system (yeast cells - 1.5 - 3 h)
Necessity for renewal
(intestine vs
hepatocyte)
State of differentiation
Tumour
How often will intesintal and hepatocyte divide
(intestinal epithelial cells - ~20 h
hepatocytes - ~1 year)
What is terminally divided cell, give examples
Cells which don’t re-enter proliferation following differentiation (neurons and cardiomyocytes)
What is consequence of abberant mitosis
Cell death
What are the key genetic properties of solid tymour cells
In addition to mutations in oncogenes and tumour suppressor genes, most solid tumours are aneuploid (abnormal chromosome number and content).
Irregegular levels of which type of protein is found in tmours
Perturbation of protein levels of cell cycle regulators is found in different tumours - abnormal mitosis
What is contact inhibition
Cells stop dividing when they reach other cells
What has been a successful means of cance therapy relating to cell division
Attacking the machinery that regulates chromosome segregation is one of the most successful anti-cancer strategies in clinical use
What are the 3 events in te cell cycle
Duplication
Division
Co-ordination
What are the 2 parts of the M phase
Mitosis (Division)
Nuclear division Cell division (cytokinesis)
What is involved in the interphase
Duplication
DNA
organelles
protein synthesis
Why is mitosis the most vulnerable period of the cell cycle
Cells are more easily killed (irradiation, heat shock, chemicals)
DNA damage can not be repaired
Gene transcription silenced
Metabolism?
Outline the phases of the cell cycle
M phase - Mitosis
Interphase:
G0 - cell cycle machinery
dismantled
G1 phase (Gap) - Decision point
S phase - Synthesis of DNA/protein
G2 phase (Gap) - Decision point
What occurs in S phase
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
What is the centrosome made up of
Consists of two centrioles (barrels of nine triplet microtubules), which are surrounded by an electron dense and protein dense amorphous cloud of pericentriolar material (PCM).
These 2 centrioles are at 90 degrees to each other… have interconnecting fibres…. mother and daughter
Outline the centrosome cycle
The centrosome cycle consists of four phases that are synchronized to cell cycle.
These include: centrosome duplication during the G1 phase and S Phase, centrosome maturation in the G2 phase, centrosome separation in the mitotic phase, and centrosome disorientation in the late mitotic phase—G1 phase.
How do microtubules get made from the centrosomes
Tubulin enters the nucleating sites in the PCM and forms tubulin ring complexes due to polymerisation of tubulin
What is the size of the naked DNA
2nm
Outline the condensation of chromatin…. when does this occur
PROPHASE:
2nm (naked)
11nm (beads on a string)
30nm (30nm chromatin fiber of packed nucleosomes)
Extented scaffhold associated form (300m)
Condensed scaffhold associated form (700nm)
Replicated (1400nm)
What are the chromosomes like in prophase
Condensed chromosomes - each consists of 2 sister chromatids, each with a kinetochore
When do the centrosomes replicate and divide into 2 poles
Late prophase
What occurs in prophase
Replicated chromosomes condense
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
What are ASTERS
What happens next
Radial microtubule arrays (ASTERS) form around each centrosome (microtubule organizing centers - MTOC)
send out microtubes in all directions
Then the radial arrays meet in the centre, forming polar microtubules (keep the MTOCs separated)
And some arrays meet the membrane, forming astral microtubuls, which anchor MTOCs to the membrane
they are in a dynamic state (constantly polymerising and depolymerising)
What occurs in metaphase
Chromosomes aligned at equator of the spindle.
What are the stages of prometaphase
Prometaphase: early prometaphase
late prometaphase
What occurs in early prometaphase
Breakdown of nuclear membrane
Spindle formation largely complete
Attachment of chromosomes to
spindle
How do chromsomes attach to the spindle
When does this occur
Attachment of chromosomes to
spindle via kinetochores (centromere region of chromosome)
in EARLY PROMETAPHASE
What occurs in late prometaphase
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
What is CENP-E
Senses the tension of the spindle attachment to the kinetochore
What occurs in anaphase
Paired chromatids separate to form two daughter chromosomes
What usually holds the sister chromatids together
Cohesin holds sister chromatids together
What occurs in anaphase A
Breakdown cohesin
Microtubules get shorter
Daughter chromosomes pulled toward opposite spindle poles
What happens in anaphase B
1-Daughter chromosomes migrate towards poles
2-Spindle poles (centrosomes) migrate apart
T/F all spindle fibres are attached to the chromatids
F…. some remain interracted with microtubes from the other pole in the centre between the poles to stabilise the structure
What occurs in telophase
Daughter chromosomes arrive at spindle
Nuclear envelope reassembles at each pole
Assembly of contractile ring
What is the contractile ring in telophase composed of
Actin and myosin
What is seen between the two daughter cells in cytokinesis. What else happens in this stage
A midbody (microtube remnant)
Also chromatin decondenses, nuclear substructured reform (not the envelope, which reforms in telophase), interphase microtubule array reassembles (not like the radial ones in mitosis)
When is there a mitotic checkpoint active
Prometaphase and metaphase
because the cells need to know that all chromatids are attached to microtubule
What does an unattached kinetochore signal to the cell
generating checkpoint signals
it tells the cell that the cell CANNOT yet proceed with mitosis
attached kinetochore then stops giving these signals
Which proteins are involved with kinetochore spindle attachment checkpoint
Requires:
CENP-E
BUB protein kinases
BUBs dissociate from kinetochore when chromosomes are properly attached to the spindle
CENP-E senses the tension of the spindle attachment to the kinetochore
When all dissociated, anaphase proceeds.
What is amphelic attachment
Each sister chromatid attached to a microtube from different pole (normal)
What is monotelic attachment
Only one microtube attached to one sister chromatid…. the other sister chromatid does not have a microtubule
Kinetochore of the unattached chromosome will produce a checkpoint signal that prevents progression to mitosis
What is syntelic attachment
When microtubules from the same pole attach to both sister chromatids (kinetochore may or may not produce checkpoint signal
Which is worse, monotelic or syntelic attachment… and why
Syntelic because the kinetochores may stop producung checkpoint signal
However, with monotelic attachment, there will be one kinetchore (i.e. the one whose sister chromatid is not attached to a microtubule) which is generating checkpoint signals, and thus the cell will not be able to proceed.
What is merotelic attachment
Attachments of 2 microtubles from opposite poles to the same sister chromatid
What happpens in syntheluc and merotelic attachment
Synthetic…. both sister chromaitds might end up at the same pole
In merotelic attachent chromosome loss at cytokinese (chromosme pulled toward both poles)
What are the two roads to aneupoidy
a) mis-attachment of microtubules to kinetochores (or no breakdown of cohesin, which holds the sister chromatids together)
b) aberrant centrosome/DNA duplication
Outline abberant centrosome/DNA duplication
Instead of the usual 2 x centrosomes, you can get a double replication such that there are 4 centrosomes.
In this case, you get weird spindle formation, and some might not even form a spindle, you could end up with 4 cells
What is the function of checkpoint kinase
Serine threonine kinase activation holds cells in G2 phase until all is ready–> inhibition leads to untimely cell transition to mitosis before the cell is ready to go into mitosis
(ps this is different from BUB protein kinases and CENP-E, which are checking for kinetochore attachment during metaphase. This is a separate checkpoint)
How do checkpoint kinase inhibitors work
Give examples of checkpoint kinase inhibitors and how they work
In addition to checkpoint kinase inhibitors, you can use taxenes and vinca alkaloids. What cancers are they used for, and what do they do
Just as a summary, we have been introduced to 2 checkpoints here.
- Kinetochore during metaphase at mitosis
- G2 checkpoint kinases (CHKE1 and CHKE2), which actually prevents cell from getting into mitosis as long as they are present
Slide 27 is kind of messed up, but it basically shows that you can use checkpoint kinase inhibitors, for either of the two checkpoints
1. If you use checkpoint kinase inhibitor you can inhibit attachment-error correction mechanism (you inhibit the checkpoint signals, make the cell think it has aligned its chromosomes, and then it’ll go into anaphase early and you induce gross chromosome mis-segregations)
- If you inhibit the checkpoint kinase (CHKE1 and CHKE2), the cell will think it is ready to progress into mitosis from G2, so there is untimely entry into mitosis, and the cancer cells will die
Taxanes and vinca alkaloids (breast and ovarian cancers)
- Alters microtubule dynamics (taxanes prevent disassembly of MTs and vinca alkaloids prevent assembly)
- Produces unattached kinetochores
- Causes long-term mitotic arrest because there will not be attachment of microtubules so kinetchores will not stop producing checkpoint signals. Remember that if they are arrested in mitosis, they are vulnerable to damage
What occurs if something goes wrong in the cell cycle,
when might each of these occur
- Cell cycle arrest
- at check points (G1 and spindle check point)
- can be temporary (i.e. following DNA repair) - Programmed cell death (apoptosis)
- DNA damage too great and cannot be repaired
- Chromosomal abnormalities
- Toxic agents
Cell cycle progression aborted and cell destroyed
What are the usual checkpoints in the cell cycle
The first checkpoint is during G1 (tells the cell to acutally enter the cycle)
Then the next checkpoint is just before mitosis, to check for DNA damage before entering mitosis (G2)- checkpoint kinases (CHKE1 and CHKE2, need to be inhibited, to allow to move from G2 to mitosis )
Then there is also a metaphase checkpoint which tests the correct sister chromatid alignment- - kinetochore checkpoint signals (need to REMOVE to allow progression beyond metaphase of mitosis) .
Tumours develop means by which they can bypass these checkpoints
How can tumours bypass the cell checkpoints
Tumours exploit the first checkpoint (i.e. G1) by hyper-activating growth factors.
G1 CHECKPOINT: The tumour cells over-express growth factors (EGF, PDGF), and the end result is induction of cells to overcome this checkpoint.
G2 CHECKPOINT: Tumours can also block the ability of the cell to sense DNA damage (e.g. p53 mutation), such that the cell bypasses this checkpoints, which can result in chromosome abnormalities.
METAPHASE CHECKPOINT: Tumour cells can also block the sister chromatid alignment checkpoint, such that the chromatids divide even when the chromosomes are not aligned
During tumorigenesis, tumours can also operate at the exit of the cell cycle. When the cells divide, they pause and enter the G0 phase (the place where they start functioning as normal cells). Tumour cells can BLOCK THIS. Once cells exit mitosis, the cycle is initiated AGAIN to continue cell division.
What usually triggers a cell to enter the cell cycle
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
How does exit from G0 occur
Exit from G0 highly regulated - requires growth factors and intracellular signalling cascades
What occurs in signalling cascades
Response to extracellular factors
Signal amplification
Signal integration
Modulation by other pathways
Regulation of divergent responses
Give example of factors responsible for moving the cell out of G0. What is the normal structure
Epidermal growth factor (EGF); Platelet-derived growth factor (PDGF)…
DIMERIC ligand
What are the receptors like for EGF and PDGF and what happens in the presence of a ligand
Respective receptors found as monomeric, inactive state
Receptor Protein Tyrosine Kinase (RPTK)
In presence of the dimeric ligand:
Receptors form dimers and they cross phosphorylate each other, and and are activated by phosphorylation.
(AA in the kinase domain at phosphorylated)
Outline the phosphorylation of AA residues in receptors
Which AAs are commonly phosphorylated
Which part is phosphorylated
The protein kinase picks up an ATP and transfers a phosphate group to the hydroxyl group (ATP to ADP)
- The serine is phosphorylated – this phosphorylation is a SIGNALLING TRIGGER
- This is REVERSED by protein phosphatases (they remove phosphate groups to restore the OH group)
- There are two types of kinase
o One type can phosphorylate serine and threonine residues
o The other type can phosphorylate tyrosine residues
How does added phosphate group change protein function
Causing a change in shape (conformation) leading to change in activity (+ve or –ve)
AND
Creating a docking site for another protein
Why are signalling cascades used
Leads to signal amplification, diversification and opportunity for regulation
