Cancer 7: External factors controlling division and behaviour

Question 1

Define cell behaviour

Answer 1

the way cells interact with their external environment and their reactions to this, particularly proliferative and motile responses of cells.

Question 2

Which external influences are detected by cells

Answer 2

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

Question 3

What are the important external factors influencing cell proliferation

Answer 3

Growth factors
Cell-cell adhesion
Cell-ECM adhesion

Question 4

What happens when a cell is in suspension vs when it hits a culture surface

Answer 4

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)

Question 5

Why does a cell spread on a culture surface

Answer 5

As it spreads, it acquires motility because it acquires a polarity (guided by a lamellipod)

Question 6

Cell spreading is a passive event, mediated by gravity. T/f

Answer 6

Cell-spreading is not a passive, gravity-dependent event

Question 7

What does cell spreading require

Answer 7

Energy is required to modulate cell adhesion and the cytoskeleton during spreading.

Question 8

What might happen if you place one cell on top of another

Answer 8

Bottom one (in contact with ECM) will spread,

one on top will bleb as it has no contact with ECM substratum

Question 9

In the presence of growth factors, how does ECM binding affect cellular proliferation?

Answer 9

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

Question 10

T/F it is the distribution rather than the amount of ECM which determines whether a cell spreads and survives

Answer 10

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

Question 11

Outline the concept of anchorage dependence

Answer 11

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

Question 12

Other than proliferation, what other cell features can be influenced by the ECM

Answer 12

Cell Phenotype can be determined by the composition of the matrix

Question 13

Give an example of the ECM affecting cell phenotype

Answer 13

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

Question 14

How do cells sense their environment, and how do we know they do this

Answer 14

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

Question 15

What are integrins

Answer 15

heterodimer complexes of a and b subunits that associate extracellularly by their “head” regions. Each of the “leg” regions spans the plasma membrane.

Question 16

Where does ligand binding occur in integrins, and what are these ligands. How many combinations of alpha and beta regions.

Answer 16

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

Question 17

Give an example of an integrin molecule which binds an ECM molecule

Answer 17

a5b1 fibronectin receptor binds arg-gly-asp (RGD)

Question 18

T/F RDG peptide sequence on the fibronectin molecule is specific to fibronectin

Answer 18

F…. peptide sequences such as RGD are found in more than one ECM molecule, e.g. RGD found in fibronectin, vitronectin, fibrinogen plus others

Question 19

What is the consequence of common peptide sequences among ECM molecules in terms of integrin binding

Answer 19

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

Question 20

T/F not all integrins link to the actin cytoskeleton via actin-binding proteins

Answer 20

T, because a6b4 integrin is found in EPITHELIAL HEMIDESMOSOMES and is linked to cytokeratin (intermediate filament) not the actin

Question 21

Outline how integrins are distributed on the cell surface

Answer 21

integrin complexes cluster to form focal adhesions (most) or hemidesmosomes (a6b4)

Question 22

What is the function of integrin clusters

Answer 22

Focal adhesion AND

SIGNAL TRANSDUCTION

Question 23

Other than ECM molecules, what else can SOME integrins bind.

Give an example of this

Answer 23

specific adhesion molecules on other cells

avb3 binds to PECAM-1(CD31) and

aIIb/b2 to ICAM-1 on endothelial cells in inflammation

Question 24

Outline a clustered integrin complex

Answer 24

Group of integrins on cell surface. Bound to ECM extracellularly and to a linker protein which then binds to actin microfilaments intracellularly

Question 25

What is ‘outside in’ integrin signalling

Answer 25

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

Question 26

How can integrin signalling be switched on and off

Answer 26

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

Question 27

In addition to the chemical detection of focal adhesions (which are essentially integrin clusters), what else can cells detect and how

Answer 27

Sense the mechanical properties of their surroundings

They detect AMOUNT OF FORCE GENERATED…..

made up of:

1. The force generated by cytoskeleton they are bound to (i.e actin for most or cytokeratin for a6b4)
2. Stiffness of the ECM

Question 28

What can integrins do within the cell and how

Answer 28

Promote signalling and actin assembly

by recruiting cytoplasmic proteins

Question 29

What is inside out integrin signaling

Answer 29

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

Question 30

Give an example of when inside out integrin signalling might be used

Answer 30

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

Question 31

When would an integrin molecule changed from a flex to an extended position.

When would the legs open in the extended position

Answer 31

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!

Question 32

What is responsible for previously described ‘contact inhibition’

Answer 32

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

Question 33

What is the relationship between the ECM and growth factor signalling

Answer 33

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

Question 34

What signals are required for a cell to proliferate efficiently

Answer 34

1. Growth factor (density dependence)

2. ECM (anchorage dependence)

Question 35

What happens if only the growth factor signalling complex, or only the integrin complex pathways are activated

What if they are both activated

Answer 35

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

Question 36

Do all cells form contact interactions when they interact?

Answer 36

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

Question 37

What happens when non-epithelial cells collide usually

Answer 37

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

Question 38

What is contact inhibition of locomotion

Answer 38

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

Question 39

Which cell types form long term cell-cell contacts

Answer 39

epithelial cells and endothelial cells, which form layers, and neurones forming synapses

Question 40

Outline types of long-term cell-cell contacts

Answer 40

cell types strongly adhere and form specific cell-cell junctions (adherens junctions, desmosomes, tight junctions, gap junctions)

In epithelial and endothelial and neurons

Question 41

How are cell- cell junctions arranged in epithelia

Answer 41

arranged as continuous belts (zonula) or as discrete spots (macula)

Question 42

What is the effect of cell-cell contact

Answer 42

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

Question 43

What occurs when there is no cell-cell junctions in terms of cell- proliferation

Answer 43

No cell-cell junctions, activated MAPK,

decreased p27KIP1, high proliferation

Question 44

What occurs when cell -cell junctions form with regard to cell proliferation

Answer 44

cell-cell junctions form, inactive MAPK,

increased p27KIP1, low proliferation

Question 45

What are cell-cell junctions dependent on

Answer 45

Ca2+ (so this will lead to cell-cell junctions, and inactivated MAPK, and low proliferation, or the reverse in the absence of calcium)

Question 46

What would be the effect on cell proliferation if an adhesion blocking antibody was introduced

Answer 46

Lack of adhesion so high proliferation

So introducing adhesion blocking antbody has same effect has LOW calcium… which is high proliferation

Question 47

Which cell-cell junctions might be involved in cell proliferation, and specifically which part

Answer 47

Adherens junctions

the beta catenin part

Question 48

Outline the structure of the adherens junctions

Answer 48

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

Question 49

What is cadherin deoendent on and what does it bind to

Answer 49

Ca2+-dependent, homophilic

cell adhesion molecule (binds to self)

Question 50

What is adenomatous polyposis coli

Answer 50

inherited colon cancer: there are a number of familial forms

Question 51

What is the APC gene product

Answer 51

a protein involved in the degradation of beta-catenin

Question 52

What usually happens in response to cadherin forming junctions at the plasma membrane

Answer 52

The APC complex will become active, and rapidly degrade beta catenin

Question 53

When might beta catenin levels rise in a cell

Answer 53

as a result of inhibition of degradation or loss of cadherin-mediated adhesion

Question 54

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)

Answer 54

beta catenin forms a complex with LEF-1

This complex enters the nucleus and influences gene expression –> PROLIFERATION

Question 55

Give examples of other adhesion-associated signalling pathways and how they influence contact-induce inhibition of proliferation

Answer 55

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.

Question 56

Does cross talk occur between cell-cell and cell-ECM adhesion,

like it did between ECM and growth factor

Answer 56

Yes it does

Question 57

What happens when cells lose behavioural restraitns

Answer 57

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)

Question 58

Aside from promoting solid tumour formation, what is an important consequence of loss of contact inhibition of locomoation for ancer progression

Answer 58

Also encourages spread

Question 59

What occurs if a gene coding for a component of a signalling pathway is mutated causing constitutive expression

Answer 59

that pathway will be permanently ‘on’.

Question 60

What molecules are proto-oncogenes

Answer 60

receptors, signalling intermediates and signalling targets (e.g. transcription factors)

Question 61

List importnat proto oncogenes

Answer 61

EGF receptor (erb)

Ras (signalling intermediate)

c-Raf (signalling intermediate)

c-Jun (transcription factor)

Question 62

Give examples of oncogenes associated with the important proto-oncogenes

Answer 62

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)

Question 63

In what percentage of cancers is Ras mutated

Answer 63

Around 30^

90% in pancreatic cancer

Question 64

What could lead to uncontrolled proliferation of tissue

Answer 64

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

Question 65

Go from slide 56

Answer 65

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