Spindle Inhibition

Question 1

What are microtubules?

Answer 1

They are key components of the cytoskeleton and are essential in eukaryotic cells.
They are long, slender, filamentous tubes comprised of alpha-tubulin heterodimers.
Tubulin heterodimers are connected by non covalent bonds. + end is associated with GTP cap and the end dynamics are faster than the – end, although both can add new heterodimer units.
There are 13 filaments.

Question 2

What are microtubules essential for?

Answer 2

Cell shape
Transport of vesicles
Play a role in cell signaling
Critical role in mitosis

Question 3

What are microtubules dynamism characterised by?

Answer 3

Rate of microtubule growth
Rate of shrinkage
Frequency of transition from growth to shrinking
Frequency of transition from shrinking to growth

Question 4

What is treadmilling?

Answer 4

When microtubule units add to one end and leave from the other.
This allows the microtubules to move while the individual heterodimers remain stationary.

Question 5

What is the role of GTP/GDP in microtubule stability?

Answer 5

Both alpha and beta units have a GTP/GDP binding site at the + end.
Within heterodimers the alpha retains a GTP tightly bounded and is non-exchangeable.
The beta + end is accessible and hydrolysis of GTP to GDP occurs at the same time or shortly after addition to the growing microtubule.
The cap normally has GTP or GDP/Pi and is stable, if this cap is lost then it is no longer stable because the ends fray.

Question 6

How is microtubule functional diversity achieved?

Answer 6

Microtubule association proteins
Soluble tubulin
Microtubule surfaces and ends 
Isotypes
Post translational modification

Question 7

What happens during interphase of mitosis?

Answer 7

Microtubules turn over slowly.
Entire network dissembles and is replaced by a new population of spindle microtubules that are 4 to 100 times more dynamic.
For at least some cells this is a result of an increase in catastrophe and a decrease in rescue rather than any change in rates of shortening and growth.

Question 8

What happens during prometaphase of mitosis?

Answer 8

Nuclear envelope is broken down.
Needs timely and correct attachment of chromosome kinetochores to the spindles.
Microtubules hunt by rapidly elongating and shrinking, probing the cytoplasm until they find the chromosome kinetochore.
They grow and shrink up to 10 micrometers until successful attachment.

Question 9

What happens in early metaphase of mitosis?

Answer 9

Complex movement of chromosomes.

Congression – movement to the equator from the metaphase plate.

Question 10

What happens in anaphase of mitosis?

Answer 10

Duplicated chromosomes separate and chromosomes move towards the two spindle poles and form two new daughter cells.

Question 11

what happens in the telophase of mitosis?

Answer 11

Duplicated chromosomes have reached new spindle poles and the cell is dividing.

Question 12

Why are microtubules targeted for cancer treatment?

Answer 12

Microtubules play a key role in organising the chromosome and cleaving to the daughter cells at the right time.
Cancer is characterised by rapid replication, targeting this key step in cell division makes a lot of sense.

Question 13

What are antimitotic drugs?

Answer 13

Surfaces of globular part of tubulin has several binding sites which allow agents to bind.
They are microtubule targeting agents which alter the dynamics and can lead to mitotic arrest and cell death.
There are two types – microtubule stabilising agents or microtubule destabilising agents.

Question 14

What are example of microtubule stabilising agents?

Answer 14

Taxane ligands

Laulimalide/ peluroside ligands

Question 15

What are examples of microtubule destabilising agents?

Answer 15

Vinca ligands
Maytensine ligands
Colchicine ligands
Prionetin ligands

Question 16

What are taxane ligands?

Answer 16

They are produced by semi-synthetic route from 10-deacetylbaccatins III.
They are used in diverse cancer therapies.
Their pocket is near the ML surface on the inside of the tubulin and acts to strengthen lateral contacts between adjacent protofilaments – leads to MT stabilisation.
Its possible paclitaxel stabilises MT via an allosteric mechanism.

Question 17

How do taxanes bind?

Answer 17

They bind poorly to the soluble pool of tubulin, but with high affinity to the site on the beta subunit along the inside of the microtubule.
Access through small gaps, or fluctuations in the microtubule lattice.
Conformational change increases lateral binding affinity.
There is one site per dimer and in principle increased polymerisation is associated with near 1:1 stoichiometry.
It is the disruption to dynamics, not the increased polymer mass, that is crucial for interruption of mitosis.

Question 18

What are laulimalide/peluroside ligands?

Answer 18

Microtubules stabilised by strengthening lateral contacts between protofilaments.
Evidence that the MSA can interact with both its host tubulin and the adjacent tubulin.

Question 19

What are vinca alkaloids?

Answer 19

They are similar to taxanes, significant side effects profile.
The vinca site is located at the plus end surface of beta tubulin.
Binding alters the surface of the + end forming a wedge.
This interferes with incorporation of new heterodimers.
The + end remains curved, and the microtubule wall cannot form.
Vinca site ligands can cause tubulin oligomers, decreasing the free tubulin pool.
At high concentrations microtubules depolymerise.
One or two ligands at the end of a microtubule will reduce dynamics by 50%
Suppression of dynamics is the key to biological activity

Question 20

What is maytansine site?

Answer 20

The site is close to the vinca site, but formed from other structures.
In growing microtubules the mayatnsine binding pocket of the + end accommodates the – end of the next alpha tubulin unit.
Incorporation of a ligand blocks this, impeding elongation.

Question 21

What is the colchicine site?

Answer 21

Colchicine isolated from autumn cross.
It has been used clinically for treating gout.
Severe toxicity at doses required for anti-tumour effects.
Lower toxicity compounds, such as combretastatin are currently in clinical trials.

Question 22

How does Colchicine bind?

Answer 22

The binding site is located between the alpha and beta units.
There is slow binding and when it binds it is irreversible.
When it binds it prevents the heterodimer being stabilised into the curved formation, during polymerisation the ends transition from curved at the tip, to straight in the body. The colchicine ligand inhibits polymerisation.
It doesn’t bind to the microtubule tip rather it binds to the soluble pool first and is then incorporated. The presence of tubulin/colchicine units slows but doesn’t prevent polymerisation.

Question 23

Why do spindle inhibitors work on selected cancers only?

Answer 23

Paclitaxel is very effective for ovarian, mammary and lung tumours.
Essentially ineffective for many other solid tumours eg colon and kidney carcinomas.

Question 24

What are the possible modes of resistance?

Answer 24

Over expression of membrane transport proteins.
Microtubule associated proteins
Post translational modification
Up and down regulation of different tubulin isotypes.

Question 25

What is the most important role for microtubule interacting drugs?

Answer 25

Changes in dynamics

Question 26

What are the expression mechanisms which exist against anticancer drugs?

Answer 26

Overexpression of multidrug resistance gene
Molecular changes in beta tubulin
Changes in apoptotic and mitosis checkpoint proteins
Changes in lipid composition
Overexpression of IL-6.