9. External Factors Controlling Cell Division

Question 1

Define cell behavior and factors that might affect it

Answer 1

Cell behaviour – term used to describe the way in which cells interact with their external environment and their reactions to this, particularly to proliferative and motile responses of the cell.

External influences:

• Chemical – i.e. hormones or growth factor.
• Physical – i.e. mechanical stress or temperature.

Main external influences to cell division:

1. growth factors
2. cell-cell adhesion
3. cell-ECM adhesion

Basic Cell Behaviour

• A cell will normally spread on a culture surface and thus acquire motility and polarity (the cell will have an obvious direction to it).
• The front is usually the motile part
• This process is an energy-dependant event (required to modulate cell adhesion and the cytoskeleton of the cell).

Question 2

Discuss the effects of cell-ECM adhesion to cell division

Answer 2

Cell-ECM Adhesion

• The cell has a greater chance of progressing into the s-phase if it spread out effectively on the adhesive patch.
• Cell require binding to ECM to be fully competent.
• The cell may apoptose If it cannot bind enough to ECM.

Anchorage Dependency:

• In suspension, cells do not significantly synthesise DNA or protein, the require to be attached to ECM (and a degree of spreading) to do this.
• Attachment to the ECM may be required for survival – Anchorage Dependency.
• Cells have receptors on their surface for ECM molecules -> receptors are linked to the cytoskeleton inside the cell -> leading to a mechanical continuity between the ECM and the cytoplasm

The Importance of Cell Spreading

• Most cells will stick to fibronectin (matrix molecule that is also found in the blood)
• If you have a defined small patch of fibronectin and put a cell on it, the cell would stick but it cannot spread and it will die of apoptosis
• If you take the same amount of fibronectin and distribute it over a number of small spots, the cell will be able to spread and it will survive and grow

Question 3

Discuss the role of integrins in cell adhesion

Answer 3

Integrins:

• Integrins are the most important of the matrix receptors
• Integrins are heterodimer complexes consisting of alpha and beta subunits
• They bind to the ECM via their heads
• Each of their tail regions cross the plasma membrane and project into the cel
• ~10 a and 8 b sub-units are known and >20 combinations.
• They recognise short, specific peptide sequences and each combination of subunits of an integrin binds to a specific sequence.

IMPORTANT – the same sequence may be found in multiple types of ECM molecule.

• Intracellularly, the integrins are linked, via actin-­‐binding proteins, to the actin cytoskeleton
• Integrins cluster to form focal adhesions (most) or hemi-desmosomes.
  
  • a6b4-integrin is found in epithelial hemi-desmosomes linked the cytokeratin (intermediate filament) cytoskeleton (exception)
  • The clusters are involved in signal transduction.
• Integrins can also bind to specific adhesion molecules found on other cell surfaces

Question 4

Discuss the signalling of ECM receptors (eg. integrins)

Answer 4

• ECM receptors (e.g. integrins) can act to transduce signals in both directions
• E.g. ECM binding to an integrin complex can stimulate the complex to produce a signal inside the cell

The signals can be from ‘outside-­‐in’

The signals can be from the ‘inside-­‐out’

“Outside-in” integrin signalling

• ECM receptors can act to transduce signals inside the cell when stimulated.A cell can receive information about its surroundings from its adhesion to the ECM which can alter the phenotype of the cell.
• E.G. In interstitial matrix (T1 collagen), mammary epithelium does not differentiate to secretory cells but in basal lamina ECM, it does organise and differentiate.
• The amount of force generated at a focal adhesion depends on both:
• The force generated by the cytoskeleton (F cell).
• The stiffness of the ECM.

“Inside-out” integrin signalling

• signals generated inside the cell can act on the integrin to alter the affinity of it.
• I.E. in inflammation or blood-clotting, this switches on adhesion of circulating leukocytes.
• The inside signals flex the receptor outwards so it has more affinity for the ligands binding.
• Integrins recruit cytoplasmic proteins which can promote signalling and actin assembly.

Integrin activation and signalling involves significant conformational changes to the complex

• The integrin on the left is in the low affinity state
• They can be switched on, into the high affinity conformation, by an inside-­‐out signal (this can be due to an external factor such as a growth factor or hormone)
• Once they bind to the matrix, you get other changes taking place
• The ligand binding also causes a change in conformation
• The legs separate and cytoplasmic signalling molecules can then bind and that binding will then allow signalling to take place -­‐ this is outside-­‐in
• When the integrins cluster, the molecules act on one another and you get signalling

Question 5

Outline the density dependence of cell division

Answer 5

• For most normal cell types, when you grow them in culture they require a surface to stick to
• As the cells become densely packed, the rate of proliferation starts to slow down
• When they fill up the space provided, they tend to stop division or it becomes very minimal
• It used to be thought that this cessation of proliferation was due to the cells running out of space
• Then an experiment was conducted where a fresh medium containing all the necessary factors for growth was spread across part of the culture surface and the cells that were exposed to these extra growth factors continued to proliferate
• This showed that it wasn’t the contact with neighbouring cells that was preventing cell division, it was actually the availability of growth factors
• The density of cells was too high so there wasn’t enough growth factor available for cell division -­‐ DENSITY-­‐DEPENDENCE OF CELL DIVISION

Question 6

Outline signals controlling proliferation tissues

Answer 6

Signals controlling proliferation of tissues

• Growth factors trigger the ERK cascade and cause cell division
• There is cross-­‐talk between ECM and growth factor signalling
• Signalling from growth factors and signalling from ECM come together to produce proliferation
• So there are two signals that interact to produce proliferation:
  
  • Growth factor (density dependence)
  • ECM (anchorage dependence)
• So growth factor receptors and integrin signalling complexes can each activate identical signalling pathways (e.g. MAPK)
• Individually, this activation is weak and/or transient
• Together, this activation is strong and sustained
• These separate pathways act synergistically (together)

Question 7

Discuss contact interactions between cells

Answer 7

Contact interactions between cells

• Short-­‐term: transient interactions between cells that do NOT form stable cell-­‐ cell junctions
• Long-­‐term: stable interactions resulting in the formation of stable cell-­‐cell junctions

Cell-­‐cell contact between non-­‐epithelial cells

• When most non-­‐epithelial cells collide, they do NOT form stable cell-­‐cell contacts
• They actually REPEL one another by paralysing motility at the contact site
• This is CONTACT INHIBITION OF LOCOMOTION
• It is responsible for preventing multi-­‐layering of cells in culture and in vivo

Cell-Cell Junctions in Epithelia

• Junctions are usually arranged as continuous belts (zonula) or discrete spots (Macula)
• When the cells made contact, there was a mutual induction of spreading
• They would form stable junctions between the cells
• This is CONTACT-­‐INDUCED SPREADING OF EPITHELIAL CELLS
• They form a coherent epithelium

Cell-cell adhesion:

+Ca2+, -adhesion-blocking-antibody -> cell-cell junctions, inactive MAPK, increased p27KIP1 and low proliferation.

When the junctions were absent:

• MAPK much more activated
• Decreased level of p27KIP1 (inhibitor of proliferation in the cell cycle)
• HIGH proliferation

When calcium was reintroduced and the junction reformed:

• MAPK inactivated
• Increased level of p27KIP1
• Low proliferation

The calcium switch caused a change in proliferation

Question 8

Discuss cadherin junctions

Answer 8

Cadherins

• Cadherins – Ca2+-dependant, homophilic cell adhesion molecules.
  
  • binds to similar molecules on the adjacent cell
• Intracellularly it binds to b-­‐catenin, which is associated with a-­‐catenin, which, in turn, links to the actin cytoskeleton
• People think b-catenin is the possible link to cell-cell adhesion.

In APC (Adenomatous Polyposis Coli), the APC gene-product is a protein involved in the breakdown of the b-catenin molecule.

Beta-Catenin

• In its normal state, b-catenin is bound to cadherin in the membrane but it can pass to the cytoplasm.
• From the cytoplasm, b-catenin can:
• Rapidly degrade when bound to APC.
• When at a high enough concentration, can bind to LEF-1 in the nucleus and alter gene transcription.
• Re-bind back onto cadherin (cannot have nuclear effects).

Mechanism for Contact Inhibition of Proliferation

• When bound to cadherin at the membrane, beta-­‐catenin is NOT available for LEF-­‐1 binding and nuclear effects
• Normally, cytoplasmic beta-­‐catenin is rapidly degraded to prevent its nuclear effects
• If beta-­‐catenin levels in the cytoplasm rise as a result of inhibition of degradation or loss of cadherin-­‐mediated adhesion, beta-­‐catenin/LEF-­‐1 complexes will enter the nucleus and influence gene expression leading to proliferation

Other cadherin-­‐associated signalling pathways are known to influence contact-­‐ induced inhibition of proliferation

• When cadherins are clustered together, you get changes in the activation of some of the small GTPases including Rac and Rho
• They are affected by GTP and they act as switches in signalling
• Changes in these small GTPases can influence proliferation
• You also get association of many growth factor receptors into cell-­‐cell contacts and that can prevent their activation
• So established cell-­‐cell contacts might actually prevent growth factor receptors from being able to respond to soluble growth factors

Cells can lose their social skills

• Under certain conditions cells will lose their behavioural restraints and, as a result, they will:
• Proliferate uncontrolably (lose density dependence of proliferation)
• Are less adherent and will multilayer (lose contact inhibition of locomotion and lose anchorage dependence)
• Epithelia break down cell-­‐cell contacts
• Not hayflick limited -­‐ they express telomerase and become immortal
• Normal cells usually form a nice monolayer
• Cancerous cell lose contact inhibition of locomotion and they will pile up on each other

Question 9

ECM adhesion in cancer

Answer 9

Cancer

• Loss of contact inhibition of locomotion à promote formation of solid tumours AND promote local invasion.
• If the gene encoding signalling pathways is mutated, the protein may be constitutively active so the pathway is ALWAYS ON.
• This is the mechanism of short-circuiting à uncontrolled proliferation because of loss of GF dependency.
• This can be seen in the picture to the left.

Oncogenes – mutant genes which promote uncontrolled proliferation.

Proto-oncogenes – normal genes corresponding to the oncogene.

E.G. receptors, signalling intermediates/targets.

Ras is mutated in ~30% of ALL cancers.

• In addition to deregulated proliferation, a major feature of cancerous tumours is the ability to spread.
• Most human cancers are carcinomas (of epithelial origin).
• To spread, the cells must break away and form a secondary tumour, distant from the primary.

Mechanism of metastasis:

1. Cell-cell adhesion broken down-regulated.
2. Cells must be motile.
3. Degradation of ECM (via MMPs) to migrate.

The degree of cell-cell adhesion is an indicator of how differentiated the primary tumour is and indicates its invasiveness and the prognosis.