Cancer 7: External factors controlling division and behaviour Flashcards
Define cell behaviour
the way cells interact with their external environment and their reactions to this, particularly proliferative and motile responses of cells.
Which external influences are detected by cells
Chemical:- hormones, growth factors, ion concs, ECM, molecules on other cells, nutrients and dissolved gas (O2/CO2) concs.
Physical:- mechanical stresses, temperature, the topography or “layout” of the ECM and other cells
What are the important external factors influencing cell proliferation
Growth factors
Cell-cell adhesion
Cell-ECM adhesion
What happens when a cell is in suspension vs when it hits a culture surface
In suspension, a cell is spherical, but when it hits a culture surface, it SPREADS
The cell sits on the culture surface and spreads (settles on the matrix surface)
Why does a cell spread on a culture surface
As it spreads, it acquires motility because it acquires a polarity (guided by a lamellipod)
Cell spreading is a passive event, mediated by gravity. T/f
Cell-spreading is not a passive, gravity-dependent event
What does cell spreading require
Energy is required to modulate cell adhesion and the cytoskeleton during spreading.
What might happen if you place one cell on top of another
Bottom one (in contact with ECM) will spread,
one on top will bleb as it has no contact with ECM substratum
In the presence of growth factors, how does ECM binding affect cellular proliferation?
It impacts hugely.
A susepended cell is less likely to enter S phase, than a cell perched on a small ECM patch, which itself is less likely to enter S phase than a cell spread on a big adhesive patch.
Cells need to be bound to ECM to be competent in responding to soluble growth factor
T/F it is the distribution rather than the amount of ECM which determines whether a cell spreads and survives
T!
If fibronectin is in a single patch, then the cell dies by apoptoosis,
if the same amount of fibronectin is distributed in small spots, the cell spreads, survives and grows
Outline the concept of anchorage dependence
Cell-ECM adhesion is critical
cells require to be attached to ECM (and a degree of spreading is required) to begin protein synthesis and proliferation (DNA synthesis)
ECM attachment may be required for cell survival
Other than proliferation, what other cell features can be influenced by the ECM
Cell Phenotype can be determined by the composition of the matrix
Give an example of the ECM affecting cell phenotype
In interstitial matrix (type 1 collagen), mammary epithelium does not differentiate to secretory cells
in basal lamina (basement membrane) matrix, mammary cells organise into “organoids” (a circle) and produce milk proteins
How do cells sense their environment, and how do we know they do this
The effects that matrix-binding can have on cell function suggest that cells can sense the composition of their environment
They have cell surface receptors LINKED TO THEIR CYTOSKELETON which bind specifically to ECM
MECHANICAL CONTINUITY between ECM and cell interior
What are integrins
heterodimer complexes of a and b subunits that associate extracellularly by their “head” regions. Each of the “leg” regions spans the plasma membrane.
Where does ligand binding occur in integrins, and what are these ligands. How many combinations of alpha and beta regions.
Ligand-binding occurs at the junction of the head regions
more than 20 combinations of a/b bind specifically to short peptide sequences of ECM proteins
Give an example of an integrin molecule which binds an ECM molecule
a5b1 fibronectin receptor binds arg-gly-asp (RGD)
T/F RDG peptide sequence on the fibronectin molecule is specific to fibronectin
F…. peptide sequences such as RGD are found in more than one ECM molecule, e.g. RGD found in fibronectin, vitronectin, fibrinogen plus others
What is the consequence of common peptide sequences among ECM molecules in terms of integrin binding
Some integrin molecules (which bind to a shared short peptide sequence) may bind more than 1 ECM molecule
But this is not the case in a5b1, the ligand of which is just fibronectin
T/F not all integrins link to the actin cytoskeleton via actin-binding proteins
T, because a6b4 integrin is found in EPITHELIAL HEMIDESMOSOMES and is linked to cytokeratin (intermediate filament) not the actin
Outline how integrins are distributed on the cell surface
integrin complexes cluster to form focal adhesions (most) or hemidesmosomes (a6b4)
What is the function of integrin clusters
Focal adhesion AND
SIGNAL TRANSDUCTION
Other than ECM molecules, what else can SOME integrins bind.
Give an example of this
specific adhesion molecules on other cells
avb3 binds to PECAM-1(CD31) and
aIIb/b2 to ICAM-1 on endothelial cells in inflammation
Outline a clustered integrin complex
Group of integrins on cell surface. Bound to ECM extracellularly and to a linker protein which then binds to actin microfilaments intracellularly
What is ‘outside in’ integrin signalling
ECM receptors (e.g. integrins) can transduce signals
I.e. ECM binds to integrin complex, stimulating complex to produce intracellular signal
So COMPOSITION OF ECM affects WHICH INTEGRIN COMPLEXES BIND (depending on the short peptide structure of ECM molecules) which can ALTER CELL PHENOTYPE
How can integrin signalling be switched on and off
Integrin molecules can have ‘flexed’ or ‘extended’ molecular shapes.
Swtiching between these positions affects their ability to bind ligands, and therefore signal.
So they can be switched on and off
Note. In the extended position, legs can be open or closed
In addition to the chemical detection of focal adhesions (which are essentially integrin clusters), what else can cells detect and how
Sense the mechanical properties of their surroundings
They detect AMOUNT OF FORCE GENERATED…..
made up of:
- The force generated by cytoskeleton they are bound to (i.e actin for most or cytokeratin for a6b4)
- Stiffness of the ECM
What can integrins do within the cell and how
Promote signalling and actin assembly
by recruiting cytoplasmic proteins
What is inside out integrin signaling
Signal generated inside cell (i.e. resulting from hormone binding to receptor) can act on integrin complex to alter AFFINITY of integrin for ECM binding
Give an example of when inside out integrin signalling might be used
in inflammation or blood-clotting, switching on adhesion of circulating leukocytes
(i.e. cytokines binding may change the affinity of leukocyte integrin molecules to endothelial cell surface receptors
When would an integrin molecule changed from a flex to an extended position.
When would the legs open in the extended position
Flex=low affinity/weak or no ligand binding
Extended= high addinity/strong ligand binding
Inside out integrin activation (signal from inside cell) switches adhesion on…. promotes extended conformation
When ECM ligand binds to this high affinity state, there is further opening of the legs. This exposes the binding site for recruitment of cytoplasmic signalling molecule, which is the OUTSIDE-IN signal!
What is responsible for previously described ‘contact inhibition’
Actually to do with compeition for external growth factors… this is the limiting factor.
Cells can be stimulated to divide at high density with flow of growth factor medium
But in normal circumstances, competiion for GF is really high when the density is high, and cells stop proliferating
so the phenomenon is called DENSITY-DEPENDENCE of cell division
What is the relationship between the ECM and growth factor signalling
Growth factor activates the ERK MAP kinase cascad, required for cyclin D to get the cell out of G0
There is cross talk between this cascade, and the ECM signalling
What signals are required for a cell to proliferate efficiently
- Growth factor (density dependence)
2. ECM (anchorage dependence)
What happens if only the growth factor signalling complex, or only the integrin complex pathways are activated
What if they are both activated
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, activation is strong and sustained
the separate signalling pathways act synergistically
Do all cells form contact interactions when they interact?
No.
short-term: transient interactions between cells which do not form stable cell-cell junctions
long term: stable interactions resulting in formation of cell-cell junctions
What happens when non-epithelial cells collide usually
they do not form stable cell-cell contacts
Repeal each other by paralysing motility at the contact site.
Promotes the formation of a motile front at another site on the cell, and moving in opposite direction
What is contact inhibition of locomotion
IN NON-EPITHELIAL CELLS
When they collide, paralyse motility at the contact site, promoting the formation of a motile front at another site on the cell, and moving off in the opposite direction.
RESPONSIBLE FOR PREVENTING MULTILAYERING OF CELLS IN CULTURE AND IN VIVO
Which cell types form long term cell-cell contacts
epithelial cells and endothelial cells, which form layers, and neurones forming synapses
Outline types of long-term cell-cell contacts
cell types strongly adhere and form specific cell-cell junctions (adherens junctions, desmosomes, tight junctions, gap junctions)
In epithelial and endothelial and neurons
How are cell- cell junctions arranged in epithelia
arranged as continuous belts (zonula) or as discrete spots (macula)
What is the effect of cell-cell contact
leads to the mutual induction of
spreading, so that the total spread area of the contacted cells is
greater than that of the sum of the two separated cells.
This could result in a stable monolayer
What occurs when there is no cell-cell junctions in terms of cell- proliferation
No cell-cell junctions, activated MAPK,
decreased p27KIP1, high proliferation
What occurs when cell -cell junctions form with regard to cell proliferation
cell-cell junctions form, inactive MAPK,
increased p27KIP1, low proliferation
What are cell-cell junctions dependent on
Ca2+ (so this will lead to cell-cell junctions, and inactivated MAPK, and low proliferation, or the reverse in the absence of calcium)
What would be the effect on cell proliferation if an adhesion blocking antibody was introduced
Lack of adhesion so high proliferation
So introducing adhesion blocking antbody has same effect has LOW calcium… which is high proliferation
Which cell-cell junctions might be involved in cell proliferation, and specifically which part
Adherens junctions
the beta catenin part
Outline the structure of the adherens junctions
Cadherin has extracellular and intracellular part.
Intraceulluar part bound to B-catenin, which is bound to a-catenin which is bound to the actin filament
What is cadherin deoendent on and what does it bind to
Ca2+-dependent, homophilic
cell adhesion molecule (binds to self)
What is adenomatous polyposis coli
inherited colon cancer: there are a number of familial forms
What is the APC gene product
a protein involved in the degradation of beta-catenin
What usually happens in response to cadherin forming junctions at the plasma membrane
The APC complex will become active, and rapidly degrade beta catenin
When might beta catenin levels rise in a cell
as a result of inhibition of degradation or loss of cadherin-mediated adhesion
What happens when beta catenin levels rise (either due to lack of adhesion, or because adhesion has occured but a faulty APC complex has not degraded the beta catenin)
beta catenin forms a complex with LEF-1
This complex enters the nucleus and influences gene expression –> PROLIFERATION
Give examples of other adhesion-associated signalling pathways and how they influence contact-induce inhibition of proliferation
Clustering of cadherins after cell-cell contact is known to alter the activation of small GTPases
e.g. Rac is activated, Rho is inhibited: this can influence proliferation.
Some growth factor receptors are associated with cell-cell junctions. This reduces their capacity to promote proliferation.
Does cross talk occur between cell-cell and cell-ECM adhesion,
like it did between ECM and growth factor
Yes it does
What happens when cells lose behavioural restraitns
proliferate uncontrollably (lose density dependence of proliferation)
are less adherent and will multilayer (lose contact inhibition of locomotion and anchorage dependence)
epithelia breakdown cell-cell contacts
not Hayflick limited, express telomerase
(i.e. cancer)
Aside from promoting solid tumour formation, what is an important consequence of loss of contact inhibition of locomoation for ancer progression
Also encourages spread
What occurs if a gene coding for a component of a signalling pathway is mutated causing constitutive expression
that pathway will be permanently ‘on’.
What molecules are proto-oncogenes
receptors, signalling intermediates and signalling targets (e.g. transcription factors)
List importnat proto oncogenes
EGF receptor (erb)
Ras (signalling intermediate)
c-Raf (signalling intermediate)
c-Jun (transcription factor)
Give examples of oncogenes associated with the important proto-oncogenes
Ras: -V12Ras (Gly12Val mutation) (signalling intermediate) -L61Ras (Gln61Leu mutation)
c-Raf:
-v-Raf (deletion of regulatory domain)
(signalling
intermediate)
c-Jun
-v-Jun (deletion of regulatory domain)
(transcription
Factor)
In what percentage of cancers is Ras mutated
Around 30^
90% in pancreatic cancer
What could lead to uncontrolled proliferation of tissue
Loss of density dependence (growth factor mediation of proliferation)
Loss of anchorage dependence (ECM mediation of proligeration)
Neither growth factor or ECM signals required to stimulate proliferation.
Mutant gene products that are CONSTITUTIVELY ACTIVE, so upstream signals are not required for the pathway to be on
Go from slide 56
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