Cell cycle and its control

Question 1

What is contact inhibition, and what happens to this process in cancer?

Answer 1

• When cells detect cells in their immediate proximity by contact, they can be inhibited in their growth and proliferation - a sort of way of autoregulating to avoid overproliferation
• This mechanism is lost in tumour cells

Question 2

In what stage of the cell cycle is the cell most vulnerable and why?

Answer 2

• Mitosis
• Because DNA damage cannot be repaired, its too late

Question 3

1) Describe the structure of centrosomes
2) Give 2 functions of centrosomes

Answer 3

1)

• 2 centrioles
• One mother and one daughter centriole
• The centrioles consist of barrels of 9 triplet microtubules
• Matrix proteins hold the mother and daughter centrioles at right angles to each other
• They will eventually form a cloud of protein complexes around them which allow the fomation of gamma tubulin ring complexes which are nucleating sites (sites where microtubules emerge from)

2)

1. MTOC (microtubule organising centre)
2. Coordinating the mitotic spindle

Question 4

Describe the key components of the life cycle of centrosomes throughout the interphase phases of the cell cycle i.e. prior to metaphase

Answer 4

• Separation of the mother and daughter centrioles in G1 phase
• In S phase, upon separation, they duplicate, the mother centriole will give rise to a daughter centriole and the daughter centriole will give rise to another mother centriole
• A cloud of protein complexes will form around the centrosomes and form gamma tubulin ring complexes which are nucleating sites (microtubule origin sites) from which microtubules begin to emerge. This must be completed before the mitotic phase

Question 5

Briefly name the 5 steps of mitosis in order

Answer 5

1. Prophase
2. Metaphase
3. Anaphase
4. Telophase
5. Cytokinesis

Question 6

Describe what occurs during the first phase in mitosis - prophase

Answer 6

• Chromatin is condensed into chromosomes…..
• The chromosomes are arranged as 2 sister chromatids joined by centromeres throughout the middle like a belt and kinetochores at the ends of the centromeres which serve as units of segregation, a point at which mitotic spindles (microtubules radiating from the centrosomes) attach
• The centrosomes have already duplicated
• The microtubules begin to radiate away from the centrosomes
• The nuclear envelope begins to break down
• Centrosomes go to opposite poles
• The centrosomes and the radiating microtubules begins to form a mitotic spindle network

Question 7

Describe the process of mitotic spindle formation

Answer 7

• Nucleation occurs emanating from the gamma tubulin nucleation sites on the MTOC (centromeres)
• Radial microtubule arrays (asters) radiate out from the centrosomes’ nucleating sites
• When the asters from centrosomes at polar ends meet, they hook to each other and are now known as polar microtubules
• Meanwhile the asters which havent yet met with those from the opposing centrosomes continue to expand the microtubule network

Question 8

Describe what occurs in metaphase, starting with early prometaphase, then going into late prometaphase

Answer 8

Early prometaphase:

• Breakdown of the nuclear envelope finalises
• Mitotic spindle formation completes
• Attachment of chromosomes to the mitotic spindle by the kinetochores - one microtubule array will attach to the kinetochore on one side (on one sister chromatid) and another microtubule array will attach on the other side (to the other sister chromatid)

Late prometaphase

• Now the chromosomes have been captured by the microtubule arrays (the mitoric spindle), the chromosomes travel along the mitotic spindle to align at the equator of the cell
• In the kinetochores there are specialised proteins that detect the attachment of the microtubule arrays onto the kinetochores…
• CENP-E detects tension in the kinetochores, when there is tension, there must be microtubules attached, so CENP-E hereby signals that metaphase has occured properly
• BUB-kinase A dissociates from the kinetochores upon microtubule attachment, to go on to signal for the start of anaphase

Question 9

Describe what occurs in anaphase, starting with anaphase A, then going into anaphase B

Answer 9

Anaphase A:

• Cohesin, which holds the 2 sister chromatids of each chromosome together is broken down, allowing easy separation of the sister chromatids
• The microtubule arrays begin to shorten, pulling the sister chromatids apart and towards the centromeres at opposite poles

Anaphase B:

• As well as microtubule array (mitotic spindle) contraction, there is also further migration of the centromeres apart to the opposite poles causing further separation of the sister chromatids
• Once the chromosomes (previously called sister chromatids) reach the opposite poles, telophase can now begin

Question 10

Describe what occurs in telophase

Answer 10

• Daughter chromosomes have arrived at the poles
• Nuclear envelope begins to reassemble at the poles
• Material condenses where the cell is going to split
• Formation of actin and myosin filament contractile rings at the separation point
• The contractile ring squeezes, you see a cleavage furrow where it is squeezing and separation will occur
• Separation almost completely occurs, it is not complete as there is a ‘midbody’ where the contractile ring acted, the separation point, its like a thin spindly attachment remnant because the daughter cells don’t completely cleave and separate yet

Question 11

Describe what occurs in cytokinesis

Answer 11

• The ‘midpoint’, which is a section at the middle betwen the 2 daughter cells and is a thin attachment between them where the actin and mysosin filament contractile rings acted to squeeze and try to separate the cells exists here initially
• In cytokinesis, this midbody is filled out with membrane and the separation is finally completed as the midpoint is completely separated so the 2 daughter cells are now completely free and independent

Question 12

What is the mitotic checkpoint that occurs during one of the phases of mitosis before the initiation of the following phase and how does this checkpoint work?

Answer 12

• Metaphase-Anaphase, there is a checkpoint where it must be ensured that the sister chromatids are attached at their kinetochores to the microtubule arrays that form the mitotic spindle
• It does this using 2 proteins found at the kinetochores…

1. CENP-E - this detects tension at the kinetochores that can only occur when the microtubule arrays are bound here
2. BUB-kinase A - this dissociates upon microtubule array attachment to the kinetochore to signal for the initiation of anaphase

Question 13

Describe the normal attachment of microtubule arrays from centrosomes onto kinetochores of chromosomes and its name, and then name and describe 3 other ways in which the attachment of the microtubule arrays from centrosomes to the kinetochores can go wrong and how this can lead to aneuploidy

Answer 13

• Amphelic (normal) attachment -
• Syntelic attachment - both kinetochores (on either side of the chromosomes on both sister chromatids) are hooked by microtubule arrays from the same centrosome. This results in the whole chromosome with both sister chromatids going to only one of the daughter cells, when there should have been segregation
• Merotelic attachment - one of the kinetochores is hooked onto a microtubule array radiating from 2 different centrosomes (on polar opposite sides), so the chromatid is being pulled in opposite directions simultaneously. This results in loss of chromosome from both daughter cells
• Monotelic attachment - only one of the kinetochores is attached to a microtubule array, the other kinetochore is unattached

Question 14

Label the diagram showing defects in the way the microtubules arrays from the centrosomes attaches to kinetochores of the chromosomes - see answers question for diagram and work it out - answers not given

Question 15

How can aneuploidy in terms of duplicated centrosomes or chromsomal aneuploidy result in aberrant cell proliferation and death?

Answer 15

• There are meant to be 2 centrosomes during mitosis (one when the cell is not about to divide)
• But sometimes you can duplicate the centrosomes and have 4 centrosomes
• This can result in aberrant cytokinesis due to abnormal microtubule array attachment to the kinetochores of the chromosomes - you’ll end up with 4 daughter cells instead of 2, so there’ll be abnormally low amount of chromosomal material within the cells
• Over-replication of the DNA where you may end up with for example 2 normal daughter cells and one without any chromosomes

Question 16

Name 2 cancer therapies that induce gross chromosome mis-segregation and then describe how they work as cancer therapies

Answer 16

1. Taxanes
2. Vinca alkaloids

• They alter microtubule dynamics and cause unnattached kinetocores
• These unattached kinetochores will signal that they are unnatached via CENP-E and BUB kinase A and ultimately through checkpoint kinases
• Normally, once the kinetochores are attached, there is signalling to induce the start of the next phase - anaphase, via these checkpoint kinases
• But you can use checkpoint kinase inhibitors that inhibit these checkpoint kinases such as serine threosine kinases
• Therefore the daughter cells will continue into anaphase despite these unattached , misattached kinetochores which will ultimately lead to chromosome mis-segregation and thus long-term mitotic failure

Question 17

1) Where in the cell cycle might there be cell growth / proliferation checkpoints?
2) How may tumours surpass these checkpoints?

Answer 17

1)

• G1 growth checkpoint - they detect the environmental stimuli such as growth factors etc and see if the contents of the cell (excluding genetic material) is sufficiently duplicated
• G2 growth checkpoint - checks for DNA damage just prior to mitosis
• Metaphase-anaphase growth checkpoint - this is to do with the signalling proteins on the kinetochores signalling if the microtubule arrays have attached to the kinetochores appropriately prior to anaphase

2)

• Tumours exploit the G1 checkpoint by hyper-expressing growth factors so you can surpass this checkpoint
• Tumours exploit the G2 checkpoint by blocking the DNA damage machinery, inducing cells to enter mitosis when they shouldn’t

Question 18

What happens at G0, and what can happen with tumours in regards G0?

Answer 18

• G0 is the exit from the cell cycle (the quiescent phase), where cells do not grow / proliferate, immediately following mitosis
• Tumours can escape this exit and continue to cycle through the cell cycle continuously, allowing hyper-proliferation

Question 19

In a signalling pathway, what might be the basic steps in a signalling pathway, specifically for growth factors

Answer 19

• Response to extracellular factors e.g. growth factors binding receptors
• This signal is transducted from the receptors down to the intracellular portion within the cytoplasm
• The signal is relayed through the cytoplasm, and is subect to modulation by other signals
• The signal is also amplified
• The signal can then diverge to multiple targets
• For growth factors, the effects it can have at the separate divergent targets include regulation of metabolic pathways, regulation of gene expression and regulate cytoskeletal changes and proliferation

Question 20

Describe in detail the steps in the response to growth factor binding their receptors that this can have on the kinase cascade pathway, giving at the start 2 examples of growth factors that can evoke this response and mentioning towards the end how this cascade response can be regulated also

Answer 20

• Epidermal growth factors (EGF) and Platelet derived growth factor (PDGF)
• Receptor protein tyrosine kinase
• These dimeric ligands will bind the monomeric receptors and effectively dimerises them
• This brings the kinase domains within the intracellular parts of the receptor closer together, resulting in cross-phosphorylation of amino acids residues within the receptors
• One type of protein kinases in the receptor will transfer a phosphate group from ATP to phosphorylate serine and threonine and another will do the same with tyrosine
• The added phosphate groups will alter protein function by changing the conformation in the proteins (+ve or -ve) or by creating a docking site for another protein (adapter proteins)
• Eventually you’ll phosphorylate other intracellular molecules such as transcription factors and so on to have their effects such as altering gene expression or phosphorylating cytoskeletal proteins or phosphorylating molecules to have metabolic effects
• REVERSE PHOSPATASES reverse the phosphorylation of proteins by these kinases

Question 21

What are the 2 types of microtubule arrays?

Answer 21

• Radial microtubule arrays (asters)
• Polar microtubule arrays - called this when the radial microtubule arrays from opposite centrosomes meet and hook together

Question 22

What is the function of protein phosphatases in the growth factor cascades?

Answer 22

• They remove the phosphate groups added by protein kinases in the growth factor signalling cascade to reverse the effects - basically going against the stimulation by the growth factor