External factors controlling division and behaviour of normal and cancerous cells

Question 1

What is important to remember about circulatory cells

Answer 1

Behave quite differently to tissue cells

Question 2

What is meant by cell behaviour

Answer 2

Cell Behaviour” is the term used to describe the way cells interact with their external environment and their reactions to this, particularly proliferative and motile responses of cells.

So it’s how the local environment affects how the cells divide and move

Question 3

What external chemical influences can be detected by cells

Answer 3

Chemical:- hormones, growth factors, ion concs, ECM, molecules on other cells, nutrients and dissolved gas (O2/CO2) concs.

Question 4

What physical external influences can be detected by cells

Answer 4

Physical:- mechanical stresses (both gross and microscopic), temperature ( we are warm blooded and so regulate temperature quite narrowly), the topography or “layout” of the ECM and other cells

Question 5

Which aspect of cell behaviour is particularly important for cancer cells

Answer 5

Cell proliferation

Question 6

What are the three best known external factors that influence cell division

Answer 6

Although all external factors may influence cell proliferation, the ones to be considered
here, in relation to cancer cell behaviour, are:-

Growth factors
Cell-cell adhesion
Cell-ECM adhesion

Although, they are all important- for example a lack of nutrients will slow down everything

Question 7

Describe what happens to cells when they are placed on culture medium (matrix)

Answer 7

It will begin to settle and spread across the surface
It will gain some sort of polarity (Ro GTPases important)
It will become motile
NOTE: this is an active process, it is not just happening because of gravity. Energy is required to modulate cell adhesion and changes in the cytoskeleton during spreading

Question 8

What is important to remember about the behaviour of cells in culture

Answer 8

Cell-spreading is not a passive, gravity-dependent event. Energy is required to modulate cell adhesion and the cytoskeleton during spreading and extension of lamellipods

Nature of spreading varies between cell types (uniform in some, non-uniform in others)

So even if you turn the culture upside down- the cells will continue to spread

Extensions of lamellipods are important in cell motility

Question 9

What will you observe on an EM when one cell sticks on top of another in cell culture

Answer 9

The top cell is blebbing as it has no contact with the ECM substratum.

The bottom cell is beginning to spread, and it has less blebbing- losing these blebs is important for the cell to be able to spread.

Question 10

Ensuring growth factors are present throughout, design an experiment where you can show the importance of ECM-adhesion in the proliferation of cells

Answer 10

a. Non-adhesive agar - cells don’t recognise agar as an ECM- they just suspend in it
Very few cells enter S phase (5%)

b. Small adhesive patch
A small proportion of cells will proliferation (30%)

c. Large adhesive patch
Almost all the cells will start proliferating (90%)

Cells require to be binding to extracellular matrix to be fully competent for responding to soluble growth factors

Question 11

Describe the importance of cell spreading for cell survival and proliferation

Answer 11

a. A small patch of fibronectin
The cell can stick but it can’t spread so it will probably die via apoptosis
b. The same amount of fibronectin spread over a larger area
The cell is able to stick AND spread so it will survive and grow
NOTE: this shows that adhesion AND spreading is important for cell survival and proliferation

Question 12

Describe anchorage dependence

Answer 12

in suspension, cells do not significantly synthesise protein or DNA
cells require to be attached to ECM (and a degree of spreading is required) to begin protein synthesis and proliferation (DNA synthesis)
attachment to ECM may be required for cell survival

These criteria are essential for responding to growth factors

Most cells need matrix

Question 13

What is important to remember about the matrix type

Answer 13

The matrix type has profound effects on the phenotype of cells. Experiments in 3-D matrix gel cultures highlight these differences.

Question 14

Describe what can happen when cells are placed in two different matrices

Answer 14

(A) in interstitial matrix (type 1 collagen), mammary epithelium does not differentiate to secretory cells; organise into a loose ball of cells- tight junctions

(B) in basal lamina (basement membrane) matrix, mammary cells organise into “organoids” and produce milk proteins. - type 4 collagen

Question 15

What does the fact that matrix binding can have an influence on cell function and proliferation suggest

Answer 15

A cell can receive information about its surroundings from its adhesion to ECM

Question 16

Describe how cells are connected to the ECM

Answer 16

Cell-ECM adhesion molecules

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 (or via linker proteins)
this arrangement means that there is mechanical continuity between ECM and the cell interior

Question 17

Describe the structure of integrins

Answer 17

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

Ligand-binding occurs at the junction of the head regions

Question 18

Compare the differences in structure between the alpha and beta chains

Answer 18

Alpha chain:
Head- contains pockets for binding of divalent cations
Legs- split at one point- joined by disulphide bonds
Short cytoplasmic tail- containing terminal COOH

Beta chain:
Legs contain cysteine rich domains
Short cytoplasmic tail has terminal COOH and can bind to talin and alpha actinin.

Question 19

Describe the family of integrins

Answer 19

more than 20 combinations of a/b known which each bind specifically to short peptide sequence on ECM proteins
For example a5b1 fibronectin receptor binds arg-gly-asp (RGD)- but it depends on the amino acids surrounding this region and also how the protein is folded.
peptide sequences such as RGD are found in more than one ECM molecule, e.g. RGD found in fibronectin, vitronectin, fibrinogen plus others
e.g. in collagens (a1b1), laminins (a6/7b1) and fibronectin (a1b1)

Question 20

Name some ECM ligands

Answer 20

Collgen
Fibronectin
Laminin
Vitronectin
Fibrinogen
Laminin-epithelial hemidesmosomes
Thrombospondin

Question 21

How many different alpha and beta subunits are there

Answer 21

10 alpha and 8 beta

There are over 20 known combinations

Question 22

What do integrins bind to intracellularly

Answer 22

most integrins link to the actin cytoskeleton via actin-binding proteins (exeption: a6b4 integrin complex found in epithelial hemidesmosomes, linked to the cytokeratin (intermediate filament) network)
integrin complexes cluster to form focal adhesions (most) or hemidesmosomes (a6b4)
these clusters are involved in signal transduction

Question 23

What else can integrins bind to

Answer 23

Some integrins also bind to specific adhesion molecules on other cells (e.g. avb3 binds to PECAM-1(CD31) and aIIbb2 to ICAM-1 on endothelial cells in inflammation)

Roles in immunity and blood clotting

Question 24

Describe how clustered integrins can form focal adhesions

Answer 24

Clustered integrin complexes in the matrix bind to cell surface receptor on cell (this is linked to a linker protein which is linked to the actin cytoskeleton).

The integrins are the cell surface receptors

Question 25

Summarise what is meant by ‘outside-in’ signalling

Answer 25

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,
i. e. “outside-in” integrin signalling

Question 26

What are the potential different configurations of integrins

Answer 26

(A-F) High resolution TEM of individual integrin complexes showing the different folded confirmations:
Knees flexed (bent) (A and B) - head facing downwards
Extended, legs closed (C and D)- head facing upwards
Extended, legs open (E and F)- head facing upwards

Question 27

Describe how integrin signalling involves conformational changes to the complex

Answer 27

Integrin complexes can adopt “flexed” and “extended” molecular confirmations. Switching between these confirmations affects their ability to bind their ligands, and their signalling. In this way, cell-ECM adhesion, and signals, can be switched on and off.

Question 28

Describe outside-in signallig of integrins

Answer 28

A cell can receive information about its surrounding via adhesion to the ECM

The ligand binds and opens the legs of the complex, allowing cytoplasmic signalling molecules to bind

Question 29

Describe how outside-in signalling can alter the phenotype of the cell

Answer 29

a cell can receive information about its surroundings from its adhesion to ECM
e.g. the composition of the ECM will determine which integrin complexes bind and which signals it receives
this can alter the phenotype of the cell

Question 30

What does the amount of force generated at the focal adhesion depend on

Answer 30

The amount of force that is generated at a focal adhesion depends on both the force generated by the cytoskeleton (F cell) and the stiffness of the ECM

Question 31

Describe how focal adhesions can sense the mechanical properties of their surroundings

Answer 31

Some proteins within the complex (linker proteins) can respond to mechanical stresses (i.e stretch)- and can open up to expose new binding sites on the proteins for different cytoplasmic signalling molecules to bind- different response from the cell.

Question 32

What is important to remember about the signalling capacity of integrins

Answer 32

Integrins have no signalling capacity
Integrins recruit cytoplasmic proteins which promote both signalling and actin assembly
Can recruit a cluster of proteins (for example Src, FAK, CAS, TAX, PAL, VIN).
FAK (focal adhesion kinase) is important in phosphorylating Src and activating its proliferative capacity.

Question 33

Describe ‘inside-out’ signalling

Answer 33

a signal generated inside the cell (e.g. as the result of hormone binding to receptor) can act on an integrin complex to alter the affinity of an integrin (i.e. alter its affinity for its ECM binding)
this is “inside-out” intergin signalling (e.g. in inflammation or blood-clotting, switching on adhesion of circulating leukocytes)- don’t want integrins on platelets to be active and adhesive all the time- you want a signal to turn them on appropiately.

Question 34

Describe how the configuration of the integrin relates to their affinity for ECM

Answer 34

Low-affinity = bent confirmation, weak or no binding to ligand

High-affinity = extended confirmation, strong binding to ligand

Question 35

Describe the relationship between inside-out signalling and outside-in signalling

Answer 35

Signal from inside the cell acts on the integrin complex to promote the switch to the extended high-affinity conformation: “inside-out” activation; switches adhesion on- from low affinity (flexed) state to high affinity (extended state)

ECM ligand binds and causes the further opening of the legs. This exposes the binding sites for the recruitment of cytoplasmic signalling molecules:

outside-in activation; signals into the cells.

Question 36

What happens to cell populations at high densities

Answer 36

At high density, cells compete for growth factors

Question 37

When cells in culture are grown in a confluent monolayer, which two theories exist to explain why their proliferation stops

Answer 37

When cells in culture form a confluent monolayer, they cease proliferating and slow down many other metabolic activities. This used to be known as contact inhibition of cell division. Another set of experiments suggest that it is competition for external growth factors and not cell-cell contact responsible:-
Density-dependence of cell division.

Question 38

Describe the experiment that explained the density-dependence of cell division

Answer 38

Grow cells in culture until they form a confluent monolayer
Inject a fresh medium of growth factor over a certain population of cells- the cells donwnstream to the injection of this growth medium will grow- so their slowing down of proliferation was dependent on the availability of growth factors and not space (contact inhibition).

Question 39

Describe the ERK MAP kinase Cascade

(reqd for cyclin D expression

Answer 39

Growth factor binds to receptor tyrosine kinase
Linked adapter protein activates ras
Ras activates raf (MAPKKK)
raf activates MEK (MAPKK)
MEK activates ERK (MAPK)

ERK translocates to the nucleus to alter gene expression and turn on proliferation.

Question 40

Summarise the cross talk between ECM and growth factor signalling

Answer 40

Both ECM signalling and growth factor signalling can cross-talk and thus act in synergy to cause proliferation of the cell
Growth factor- ERK MAPK Cascade
ECM- cdc43-PAK pathway, protein cluser (src,FAK ETC) pathway can act on ERK MAPK cascade to cause cell proliferation.

Question 41

What are the two signals contributing to the proliferation of cells

Answer 41

Growth factor (density dependence)
ECM (anchorage dependence)

Both signals needed for efficient stimulation of proliferation.

Question 42

Describe the interaction between ECM and growth factor signalling

Answer 42

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 43

Summarise the short-term and long-term contact inhibition between cells

Answer 43

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 44

Describe cell-cell contact between non-epithelial cells

Answer 44

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, promoting the formation of a motile front at another site on the cell, and moving off in the opposite direction.

This is contact inhibition of locomotion and is responsible for preventing multilayering of cells in culture and in vivo.

Question 45

Describe long-term cell-cell contacts

Answer 45

Upon contact, some cell types strongly adhere and form specific cell-cell junctions (adherens junctions, desmosomes, tight junctions, gap junctions) (see 1st year MCD lecture).

This is true of epithelial cells and endothelial cells, which form layers, and neurones forming synapses.

Question 46

What are the different types of cell-cell junctions in epithelia

Answer 46

Junctions are usually arranged as continuous belts (zonula) or as discrete spots (macula)

Question 47

What do adherens junctions consist of

Answer 47

Desmosomes
Hemidesmosomes
Focal contact
Adhesion belt

Question 48

Describe the contact induced spreading of epithelial cells

Answer 48

Contact between epithelial cells 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.
Spreading is due to the polymerisation of actin at the site of contact.

Question 49

What are the effects of low calcium levels on an epithelium

Answer 49

Many cell-cell junctions are calcium dependent
In the absence of calcium/low calcium, the junctions will break down
This leads to:
· Increased MAPK activation
· Decrease activity of p27KIP1 (Cdk inhibitor)
· INCREASED PROLIFERATION

When calcium returned to normal and the junctions were reformed:
· Decreased MAPK activation
· Increased activity of p27KIP1 (Cdk inhibitor)
· DECREASED PROLIFERATION

Question 50

What are the effects of adhesion blocking antibodies on cell proliferation

Answer 50

The same results as Ca2+ were achieved were achieved
This showed that cell-adhesion affects proliferation
Low antibody, high Ca2+= low proliferation

Question 51

Describe the structure of an adherens junction

Answer 51

There is a cadherin domain that is transmembrane and projects extracellularly
Cadherins are homophilic and associate with similar structures on adjacent cells
Intracellularly, the cadherin is bound to b-catenin, which is bound to a-catenin, which, in turn, is bound to the actin cytoskeleton (via Fmn)

Question 52

What type of molecule is Cadherin

Answer 52

(Ca2+-dependent, homophilic

cell adhesion molecule)

Question 53

What is important to consider about Beta-catenin

Answer 53

Is b-catenin
the link between
cell-cell adhesion
and proliferation ?

Question 54

What is adenomatous polyposis coli

Answer 54

inherited colon cancer: there are a number of familial forms)
The APC gene-product is a protein involved in the degradation of the junction-associated molecule, b-catenin.
This is faulty in these patients- so B-cantenin is associated with LEF1 instead- which forms a transcription factor which leads to cell proliferation.

Question 55

Describe B-cantenin dynamics in cells

Answer 55

Sequestered
(bound to cadherin forming junctions at plasma membrane)

If these junctions are degraded, you will get release of beta-cantenin into the cytoplasm:
If the APC complex is active, APC will bind to B-cantenin-leading to its rapid degradation
If the APC complex is inactive (APC)- B-cantenin will bind to LEF-1- the resulting complex can act as a transcription factor- gene transcription leading to cell proliferation.

You rarely get high concentrations of beta-cantenin in the cytoplasm.

Question 56

Describe the mechanism for the contact inhibition of proliferation

Answer 56

when bound to cadherin at the membrane, b-catenin not available for LEF-1 binding and nuclear effects
normally, cytoplasmic b-catenin rapidly degraded
if b-catenin cytoplasmic levels rise as a result of inhibition of degradation or loss of cadherin-mediated adhesion, b-catenin/LEF-1 complex enters nucleus and influences gene expression, leading to proliferation.

Question 57

Describe some other cadherin-associated signalling pathways that are known to influence contact-induced inhibition of proliferation

Answer 57

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 58

Describe the consequences of cells losing their ‘social skills’

Answer 58

Under certain conditions, cells lose their behavioural restraints. As a result, they will:-
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 59

What are the consequences of a loss of contact inhibition for the progression of cancer

Answer 59

Cells will form multi-layers- solid tumours

Cells will invade the local tissues.

Question 60

What is important to remember about the components of signalling pathways

Answer 60

many components of signal transduction pathways are proto-oncogenes
so these can become oncogenes- leading to a loss of control- cancer

Question 61

Describe how cells may lose control of their proliferation

Answer 61

if the gene coding for a component of a signalling pathway is mutated so that the protein is constitutively active, that pathway will be permanently ‘on’.
receptors, signalling intermediates and signalling targets (e.g. transcription factors) are proto-oncogenes
this is the mechanism of short-circuiting leading to uncontrolled proliferation as a result of loss of growth factor dependence etc.

Question 62

Define oncogene

Answer 62

Oncogene:

mutant gene which promotes uncontrolled cell proliferation

Question 63

Define proto-oncogene

Answer 63

Proto-oncogene:

normal cellular gene corresponding to the oncogene

Question 64

Match some proto-oncogenes to their oncogenes

Answer 64

Protoncogene- EGF receptor
(Erb)
Oncogene- none

Protooncogene- Ras (signalling intermediate)
Oncogene- V12Ras (Gly12Val mutation)
Oncogene- L61Ras (Gln61Leu mutation)

Protooncogene- c-Raf (signalling intermediate)
Oncogene- v-Raf (deletion of regulatory domain)

Protooncogene- c-Jun (transcription factor)
Oncogene- v-Jun (deletion of regulatory domain)

Question 65

Describe the importance of Ras

Answer 65

Ras is mutated in ~30 % of all cancers
Most in pancreatic- 90%
None in  papillary (thyroid)
Colorectal
Kidney
Seminoma  						43
Melanoma 						13
Bladder 						10
Liver 							30
Kidney  							10
Myelodysplastic syndrome  				40
Acute myelogenous leukemia 				30

Non-small-cell lung cancer (adenocarcinoma)  		33
Colorectal  						44
Pancreas  						90
Thyroid 
	Follicular  					53
	Undifferentiated papillary 			60

Question 66

Summarise the uncontrolled proliferation of tissue cells

Answer 66

= mutant gene products that are constitutively active, therefore the upstream signals are not required for the pathway to be “on”. (no need for ligand activation)

Neither growth factor (density dependence lost) or ECM signals (anchorage dependence lost) required to stimulate proliferation.

Question 67

What are benign tumours surrounded by

Answer 67

A fibrous connective-tissue capsule

Question 68

Summarise local invasion and metastasis

Answer 68

in addition to deregulated proliferation, a major feature of cancerous tumours is their ability to spread
most human cancers are carcinomas (i.e. of epithelial origin)
in order to spread to other sites (metastasis), cells must break away from the primary tumour, travel to a blood or lymph vessel, enter the vessel, lodge at a distant site, leave the vessel, and ultimately establish a secondary tumour

Question 69

How does a primary carcinoma cell metastasise

Answer 69

cell-cell adhesion must be down-regulated (e.g. cadherin levels reduced)
the cells must be motile- need to lose contact inhibition of locomotion to metastasise successfully
degradation of ECM must take place; matrix metaloproteinase (MMP) levels increased in order to migrate through basal lamina and interstitial ECM
the degree of carcinoma cell-cell adhesion is an indicator of how differentiated the primary tumour is, and indicates its invasiveness and the prognosis

Question 70

Outline the progression of a carcinoma to metastasis

Answer 70

Normal
Dysplasia
Carcinoma in situ
Malignant carcinoma
Break through basal lamina
Invade capillary (1 in 1000 will survive travel in blood)
adhere to capillary wall
escape from capillary (extravasation)
proliferate to form metastasis in distant tissue (i.e liver)

Question 71

What is important to remember about cells in suspension

Answer 71

• in suspension, cells do not significantly synthesise protein or DNA
• cells require to be attached to ECM (and a degree of spreading is required) to begin protein synthesis and proliferation (DNA synthesis)
• attachment to ECM may be required for survival (e.g. epithelia, endothelia)