7. External factors controlling division and behaviour of normal and cancerous cells Flashcards
What is the term ‘cell behaviour’ used to describe?
The way cells interact with their external environment and their reactions to this (particularly proliferative and motile responses of cells)
What external influences do cells detect?
Chemical influences • hormones • growth factors • ion concentration • ECM (density, composition) • molecules on other cells • nutrients • dissolved gas concentrations
Physical influences
• mechanical stresses
• temperature
• topography of the ECM
What external factors can influence cell division (in relation to cancer)?
- Growth factors
- Cell-cell adhesion
- Cell-ECM adhesion
How does a cell change when it is moved from a suspension to a culture surface?
- Spherical => spreads
- Gains motility as it acquires a polarity (guided by lamellipodia and filopodia)
- Energy required to modulate cell adhesion and the cytoskeleton during spreading (not gravity dependent)
- When the culture is turned upside down, the cells will stills spread
What is blebbing and when can it happen?
- Cell detaches its cytoskeleton from the membrane
- Causes membrane to swell into spherical bubbles
- Can happen if there is a cell on top of another, so it has no contact with the ECM substratum
What is necessary for a cell to be able to respond to growth factors (in terms of ECM)?
- Cells have to be adhered to matrix in order to respond to external GFs => proliferation
- Cell has to be able to fully spread out as well
- ‘Anchorage dependence’
How does cell survival differ in suspension and attachment to ECM?
- Suspension - do not significantly synthesise protein or DNA (no S phase)
- ECM - combined with spreading, allows protein synthesis and proliferation to occur
How is the cell attached to the ECM?
- Cells have receptors which bind specifically to ECM molecules
- Linkage at cytoplasmic domains allows mechanical continuity between cell interior and ECM
Describe the results in a test where mammary epithelial cells were mixed with gels made up of 2 type of ECM?
Interstitial matrix (type 1 collagen)
• cells cluster together (loose and undifferentiated)
• hormones present in culture but no effect on the cells
• not secretory cells
Basal lamina matrix gel
- cells form a spherical cyst (hollow ball)
- these organoids switch on the production of milk proteins
Describe the structure of integrins
(one of the groups of cell-ECM adhesion complexes)
• Heterodimer complexes of alpha and beta subunits
• Associate extracellularly by their ‘head’ region
• Beta unit has a slightly longrr cytoplasmic tail
• Lingand-binding (to ECM) occurs at the junction of the ‘head’ regions
• Most are linked to the actin cytoskeleton via actin-binding proteins
What is the most important ECM receptor?
Integrin
How many different known combinations of integrin are there?
- 20 combinations
* About 10 alpha chains and 8 beta chains known
What do integrins recognise?
• Each combination specifically binds a particular, short, peptide sequence within a matrix molecule
• Such peptide sequences can be found in more than one ECM molecule
e.g. a5b1 receptor binds to arg-gly-asp (RGD), found in fibronectin, vitronectin, fibrinogen etc.
Are integrins rigid or flexible?
- Flexible
- Can adopt different combinations (folded, intermediate or extended)
- Important for its function
What is the only integrin that we know of that isn’t associated with the actin cytoskeleton?
a6b4 integrin complex found in epithelial hemidesmosomes, linked to the cytokeratin (intermediate filament) network
What types of clusters do integrins form and what are they involved in?
- Focal adhesions (most)
- Hemidesmosomes (a6b4)
- Involved in signal transduction
Apart from epithelial cells, what other cells can integrins bind to (and via which protein)?
- White blood cells - PECAM-1 (CD31)
* Endothelial cells in inflammation - ICAM-1
What are focal adhesions?
- Large, dynamic protein complexes through which the cytoskeleton of a cell connects to the ECM
- Found at the ends of bundles of filamentous actin (microfilaments of the cytoskeleton)
What is outside-in signalling?
- Cell binds to the matrix via the integrin
- Stimulates an intracellular signal
- Cell can receive information about its surroundings from this
- Composition of ECM determines which integrin complexes bind and which signals it receives
- This can alter the phenotype of the cell
- However, not all cells express the same integrins
How do focal adhesions sense the mechanical properties of their surroundings, and how do molecules take advantage of different forces?
• Amount of force generated at focal adhesions depends on:
- the force generated by the cytoskeleton (F cell)
- stiffness of the ECM
• Some of the ECM can be coiled, and when it is stretched, it is exposed to allow other molecules to bind for specific function
Do integrins have any enzymatic activity and how do they signal?
• No enzymatic activity
• Therefore, when integrins cluster, they recruit:
- signalling molecules
- molecules that associate with the actin cytoskeleton
How do integrins change configuration when switched on and off?
- On - extended
* Off - flexed/bent
What is inside-out signalling and when is it used?
• Signal generated inside the cell (due to hormone binding to receptor)
• Affinity of integrin is altered - extended conformation
e.g. inflammation, blood clotting, adhesion of circulating leukocytes
• Allows cells to be able to stick when necessary e.g. over-adhesion of platelets could cause clots
(• Ligand binding can then cause a further change to the molecule)
When normal tissues cells are plated in a culture dish, when do they stop proliferating and why??
Once the culture surface area is covered, forming a confluent monolayer
• Contact inhibition
• Competition for external growth factors
How does density affect cell proliferation?
- Cells can still divide at a high density
- However, the higher the density, the lower the rate of proliferation
- Cell-cell contact is not responsible … density-dependence is
Describe the ERK MAP kinase cascade?
Cascade required for cyclin D expression
• GF binds to receptor tyrosine kinase
• Causes adaptor molecule to bind and recruit Ras protein
• Ras activates RAF
=> MEK
=> ERK
(various levels of MAP-kinases)
• These can influence gene expression and stimulate proliferation
• GF is not enough, as cells need to be bound to matrix
Outline the mechanism of anchorage dependence coinciding with GF signals
- Integrin signalling complexes and GF receptors can activate identical signalling pathways e.g. MAPK, in different ways
- Individually, this is weak/transient
- Together, activation is strong and sustained
- The separate signalling pathways act synergistically
ECM influence on the cell is associated with ‘anchorage dependence’, as GF influence is associated with…
Density dependence
How does the time of contact interaction between cells have an affect?
Short-term
• transient interactions between cells => do not form stable cell-cell junctions
• when most cells collide, they actually repel one another by paralysing motility at the contact site
• this promotes the formation of a motile front at another site and movement in the opposite direction (contact inhibition of locomotion)
• responsible for preventing multi-layering of cells
Long term
• stable interactions resulting in formation of cell-cell junctions (adherens, desmosomes, tight, gap)
• true of epithelial cells forming layers, and neurones forming synapses
What are the 2 ways cell-cell junctions are arranged?
- Zonula - continuous belts
* Macula - discrete spots
Give an example of when contact-induced spreading is used
- Contact between epithelial cells leads to the mutual induction of spreading
- Done so the total spread area of the contact cell is greater than the sum of the 2 separated cells
- Results in a stable monolayer
What are cell-cell junctions dependent on, and what happens if they don’t form?
- Calcium needed for formation
- If not, MAPK is activated
- p27KIP1 is decreased
- This leads to high proliferation
What happens is calcium is added once MAPK is activated?
- MAPK is deactivated
- p27KIP1 is increased
- Low proliferation
What can you use to actively block adhesion?
- Add adhesion blocking antibodies instead of calcium
* They would bind to the cell adhesion molecules
What is cadherin and what is it associated with intracellularly?
- Calcium-dependent, homophilic cell adhesion molecules
* Cytoplasmic tail is associated with beta-catenin, alpha-catenin and the actin cytoskeleton (in order)
What is APC and how can it cause colon cancer?
• APC = adenomatous polyposis coli
- tumour suppressor gene
• It is a protein involved in the degradation of the junction-associated molecule: beta-catenin
• If mutated, it reduces the degradation of beta-catenin
• In the cytoplasm, it binds with LEF-1, enters the nucleus and alters cell proliferation
• If degradation is reduced, this leads to cell proliferation
How can beta-catenin be important in a mechanism for contact inhibition?
- When bound to cadherin at the membrane, beta-catenin is not available for LEF-1
- Therefore, its nuclear effects of proliferation aren’t achieved
What effect does the clustering of cadherins, after cell-cell contact, have on GTPases?
Alters their activation
e.g. Rac activation, Rho inhibition => proliferation influenced
Under certain conditions, cells can lose their behavioural restraints. What does this involve?
• Uncontrollable proliferation
• Less adherent and will multiplayer (lose contact inhibition of locomotion and anchorage dependence)
- touch for prolonged periods of time
- promotes formation of solid tumours
• Epithelial breakdown cell-cell contacts
• Not Hayflick limited, express telomerase i.e. cancer
If a mutant gene forms products that are constitutively active, what does this mean for upstream signals?
- Pathway is always switched on
- Upstream signals e.g. GFs and integrin complex signalling, is not needed
- Density dependence and anchorage dependence is lost
- Allows cell to proliferate without any limitations
How does a primary carcinoma cell metastasise?
- Cell-cell adhesion down-regulated
- Cell must be motile
- Degradation of ECM - matrix metalloproteinase (MMP) levels increase in order to migrate through basal lamina and interstitial ECM
- Cells can break away and travel to other sites, to form a secondary tumour
What can be used as an indicator of tumour differentiation, and how does this affect its likeliness to spread?
- Degree of cell-cell adhesion
- Indicates its invasiveness and prognosis
- Less differentiated
- More likely to spread
