Define cell behaviour.
– the way cells interact with their external environment and their reactions to this, in cancer it’s particularly the proliferative and motile responses of cells.
What three factors influence cell proliferation?
- Growth factors
- Cell-cell adhesion
- Cell-ECM adhesion.
Explain how attachment to the ECM is necessary for cell survival.
Cells require to be attached to ECM (and a degree of spreading is required) to begin protein synthesis and proliferation.
- Anchorage dependence: attachment to ECM is required for survival, if you left cells in suspension they would eventually die.
- Anoikis is a form of programmed cell death which is induced by anchorage-dependent cells detaching from the surrounding extracellular matrix
- Cells need to bind to matrix in order to respond to the soluble growth factors present and progress cell cycle.
- Cells have receptors on their cell surface which bind specifically to ECM molecules. These molecules are often linked, at their cytoplasmic domains, to the cytoskeleton - this arrangement means that there is mechanical continuity between ECM and the cell interior
- E.g. integrins.
What are integrins?
Important cell-ECM adhesion receptors.
- They are heterodimer receptor complexes of alpha and beta subunits (which both span membrane) that associate extracellularly by their “head” regions.
- Each of the “tail” regions spans the plasma membrane.
- They recognise short, specific peptide sequences that are found in ECM molecules (e.g. a5b1 fibronectin receptor binds arg-gly-asp (RGD))
- More than 20 combinations of a/b known – different combinations will dictate the ligand it binds.
- Once ligand binds, they cluster to form complexes called focal contacts (in most integrins) or hemidesmosomes (in a6b4). Usually these are linked to ends of actin cytoskeleton via actin binding proteins.
- These are involved in signal transduction.
- Integrins also bind to specific adhesion molecules on some cells (e.g. avb3 binds to PECAM-1(CD31) and a11b2 to ICAM-1 on endothelial cells in inflammation)
- Cytoplasmic subunit has no catalytic activity but is important in signal transduction – integrins can bind ECM and cause signal inside cell
What are the two methods of integrin signal transduction?
- Outside in signalling
2. Inside out signalling
Explain the mechanism of outside-in integrin signalling.
- ECM receptors (e.g. integrins) can act to transduce signals.
e. g. ECM binding to an integrin complex can stimulate the complex to produce a signal inside the cell,
- ECM receptors (e.g. integrins) can act to transduce signals.
- A cell can receive info about its surroundings by adhesion to ECM, i.e. the composition which will determine the integrins it binds and therefore it can alter phenotype of the cell.
- The ligand binding causes legs of complex to open to allow cytoplasmic signalling by recruiting cytoplasmic proteins which will promote signalling and actin assembly.
Explain the mechanism of inside-out signalling.
- An intracellular signal (e.g. as the result of hormone binding to receptor) causes conformational changes on receptor changing its affinity for ligand. (i.e. alter its affinity for its ECM binding).
- Important in inflammation or blood-clotting, switching on adhesion of circulating leukocytes) – low affinity signalling.
- Inside out signalling will extend the flexed complex of receptor which increases affinity
How can matrix composition affect cells? Explain what density dependence is?
- The type of matrix can have significant effects on the cell phenotype.
- Experiment in 3-D matrix culture: grew mammary epithelium in two matrix environments: collagen (no milk proteins) and laminin (milk proteins).
- Collagen – cells formed clumps, no differentiation to secretory cells.
- Laminin – cells polarised, formed apical and basal domains and produced milk proteins.
- Cells compete for growth factors, density dependence: cells grow and consume their nutrients until they run out where they enter a quiescent state. If you then pump nutrients into this area the cells will still be able/competent to divide. This shows that when cells become quiescent it is due to competition between other cells for limited growth factors.
- This wasn’t contact inhibition as it wasn’t caused by cell-cell contact.
How does anchorage dependence affect growth factor signalling?
- There is cross talk between ECM/integrin and growth factor signalling:- both will stimulate the same signalling cascades (such as MAPK).
- -This is needed as individually their actions are very weak or transient but when combined their activation is strong and sustained – synergistic, and proliferation then occurs.
- Therefore anchorage is required for cell proliferation.
Explain how long-term, stable interactions between cells can affect proliferation.
Long term, stable interactions resulting in the formation of cell-cell junctions:
– Upon contact, some cell types strongly adhere and form specific cell-cell junctions (adherens junctions, desmosomes, tight junctions, gap junctions)
– This is true of epithelial cells and endothelial cells, which form layers, and neurones forming synapses
– When epithelial cells contact, they induce spreading mutually so that the space taken up by two junctioned cells is bigger than two separate cells. This could form a stable monolayer.
– Cell adhesion affects cell proliferation:
o No Cell-cell junctions – activation of MAPK, high proliferation
o Cell-cell junctions – low proliferation, no activation of MAPK
How is adherens junction linked to cell proliferation?
- Cadherins (calcium dependant, homophilic proteins) are important molecules in the adherens junction who interact with catenins which then interact with actin cytoskeleton
- The beta catenin link to cadherin and alpha cadherin is thought to affect proliferation
- APC: Adenomatous polyposis coli, an inherited colon cancer. The APC gene product protein is involved in degradation of b-catenin. Mutations in APC leads to less degradation of beta catenin – overproliferation.
- The WNT-1 oncogene is activated causing the degradation complex of beta-catenin to be impaired so beta-catenin, a transcriptional activator to act on target genes which promote proliferation.
- When bound to cadherin at the membrane, b-catenin not available for LEF-1 binding and nuclear activation.
- If b-catenin cytoplasmic levels rise as a result of loss of cadherin-mediated adhesion, b-catenin/LEF-1 complex enters nucleus and influences gene expression of c-myc etc, leading to over proliferation and cells multi-layer on top of themselves (too much growth - polyps).
