External factors controlling division and behaviour of normal and cancerous cells

Question 1

What are three external factors that influence cell division and are important in cancer cell behaviour?

Answer 1

Growth factors
Cell-cell adhesion
ECM-cell adhesion

Question 2

Describe what happens to a cell when it is placed on a culture medium. What kind of process is this?

Answer 2

• It will begin to settle and spread across the surface
• It will gain some sort of polarity
• It will become motile

NOTE: this is an active process (not gravity-dependent)

Question 3

Describe the degree of proliferation/probability entering S-phase for cells that have:
a. suspended in non-adhesive agar
b. perched on small adhesive patch
c. spread on a big adhesive patch
Note that in each case growth factors were sufficiently present.
Hence, what can be concluded?

Answer 3

a. Very few cells enter S phase
b. A small proportion of cells will enter S phase
c. Almost all the cells will start proliferating/enter S phase

Cells require binding to ECM to be fully competent in responding to soluble growth factors so that the cell can begin protein synthesis and proliferation (DNA synthesis).

Question 4

What is the difference in proliferation when a cell is placed on:
a. a defined amount of fibronectin in a single patch
b. the same amount of fibronectin distributed in small spots
What does this show?

Answer 4

a. cell dies by apoptosis
b. cell spreads, survives and grows

This shows that adhesion AND spreading is important for cell survival and proliferation

Question 5

What is the term given to the requirement of ECM attachment for cell survival and growth?

Answer 5

anchorage dependence

Question 6

Use an example to explain why cell phenotype can be determined by the composition of the matrix the cell is attached to.

Answer 6

Mammary epithelial cells cultured in:
(A) interstitial matrix (type 1 collagen) => the cells do not differentiate to secretory cells

(B) in basal lamina (basement membrane) matrix (where they normally occur) => the cells organise into “organoids” and produce milk proteins.

Question 7

Describe the structure of integrins.

Where does ligand-binding occur?

Answer 7

• There are heterodimer complexes of alpha and beta subunits
• They associate extracellularly via their head and each of the tail regions spans the plasma membrane
• Ligand-binding occurs at the junction of the head regions

Question 8

How many different combinations of alpha/beta subunits are their?

Answer 8

more than 20 combinations

Question 9

What do the extracellular parts of integrins bind to? Give an example.

What can some integrins also bind to?

Answer 9

Short, specific peptide sequences
e.g. arg-gly-asp (RGD sequence) found in fibronectin, vitronectin, fibrinogen plus others

Some integrins also bind to specific adhesion molecules on other cells

Question 10

What do most integrins bind to intracellularly, and via what? Hence what?

Answer 10

Actin cytoskeleton (via actin-binding proteins)
Hence there is mechanical continuity between the ECM and the cell interior

Question 11

State the integrin that is an exception in that it doesn’t bind intracellularly to actin.

Answer 11

alpha6beta4 integrin complex found in epithelial hemidesmosomes, linked to the cytokeratin

Question 12

Integrin complexes cluster to form what?

Answer 12

Focal adhesions (most) or hemidesmosomes (a6b4)

Question 13

What is the other important purpose of integrins other than cell adhesion?

Answer 13

signal transduction

Question 14

State the different conformations of the integrin complex. How does this determine their ability to bind their ligands, and their signalling?

Answer 14

Integrin complexes can adopt “flexed” and “extended” molecular confirmations.
Flexed = knees of legs are bent (low affinity for ECM => signal OFF)
Extended = legs are straight and open (high affinity for ECM => signal ON)

Question 15

Describe outside-in signalling of integrins.

Answer 15

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 16

Describe inside-out signalling of integrins. Give an example of its importance.

Answer 16

Growth factors or hormones can generate signals inside the cell, which can act on the integrin complex and alter its affinity for ECM binding (by changing its conformation)
e.g. important in bloodclotting and inflammation

Question 17

When cells in culture form a confluent monolayer, they cease proliferating and slow down many other metabolic activities. What did this used to be explained by? Experiments have suggested what instead as being responsible for this?

Answer 17

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 18

What two signals work synergistically to trigger proliferation in cells?

Answer 18

• ECM signalling (anchorage dependence)
• growth factor signalling (density dependence)

Both activate identical signalling pathways (e.g. MAPK); individually, this activation is weak and/or transient but together, activation is strong and sustained.

Question 19

What happens to most non-epithelial cells when they make contact with each other? What is this called and why is it important?

Answer 19

They will move away from each other (transient interaction)
The motility on the side that made contact will become paralysed meaning that the cells can then move away from each other

This is called CONTACT INHIBITION OF LOCOMOTION
Important as it is responsible for preventing multi-layering of cells.

Question 20

Which types of cells form stable cell-cell junctions when they come into contact?

Answer 20

Epithelial cells
Endothelial cells
Neurones
Myocardium

Question 21

What are the two types of cell-cell junction?

Answer 21

Zonulae (belts)

Maculae (spots)

Question 22

What happens to epithelial cells when they come into contact with one another? What could this result in?

Answer 22

Contact between epithelial cells leads to mutual induction of spreading, so that the total spread area of the contacted cells is greater than the sum of the two separated cells. This could result in a stable monolayer.

Question 23

How does cell-cell adhesion affect cell proliferation?

Answer 23

No cell-cell junctions form => activated MAPK,
decreased p27KIP1 => HIGH proliferation

Cell-cell junctions form => inactive MAPK,
increased p27KIP1 => LOW proliferation

*Note that p27KIP1 is an inhibitor of cell proliferation.

Question 24

The formation of cell-cell adhesions is dependent on what?

Answer 24

Ca2+ dependent

i.e. low Ca2+ => high proliferation; high Ca2+ => low proliferation

Question 25

How can you prove that it is the formation of the cell-cell adhesions and not the Ca2+ directly which determines the level of proliferation?

Answer 25

Have the cells be in the presence/absence of an adhesion blocking antibody as well as the Ca2+.
=> same results would be achieved

Question 26

Describe the characteristics/structure of an adherens junction, namely a cadherin - state the different domains and what they bind to.

Answer 26

• Ca2+-dependent
• homophilic (associate with similar structures on adjacent cells)
• transmembrane and projects extracellularly
• Intracellularly, the cadherin is bound to beta-catenin, which is bound to alpha-catenin, which, in turn, is bound to the actin cytoskeleton

Question 27

Describe the action of beta-catenin when it isn’t sequestered by cadherin (i.e. bound to cadherin forming junctions at plasma membrane)

Answer 27

Normally, beta-catenin free in the cytoplasm is rapidly degraded by APC so it doesn’t tend to achieve high concentrations.

Otherwise, free beta-catenin can bind to LEF-1 to form a transcription factor to regulate gene expression and promote proliferation

Question 28

Explain how the APC gene mutation causes adenomatous polyposis coli (type of inherited colon cancer)

Answer 28

The APC mutation means that the APC protein can no longer degrade beta-catenin as efficiently
So beta-catenin accumulates in the cytoplasm, associates with LEF-1 and triggers abnormally increased proliferation

Question 29

What is contact inhibition of proliferation?

Answer 29

When bound to cadherin at the plasma membrane, beta-catenin is NOT available to bind to LEF-1 and cause nuclear effects => decreased/inhibited proliferation.

Hence, cytoplasmic levels of beta-catenin can rise if there is:

• Inhibition of degradation
• Loss of cadherin-mediated adhesion

Question 30

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

Answer 30

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 31

Describe ways in which cells can lose their social skills.

Answer 31

• Proliferate uncontrollably (loss of density dependence)
• Become less adherent and multi-layer (loss of contact inhibition of locomotion and loss of anchorage dependence)
• Epithelia break down cell-cell contacts
• Not hayflick limited (express telomerase and become immortal)

Question 32

Other than promoting the formation of solid tumours, what is an important consequence of loss of contact inhibition of locomotion for the progression of cancer?

Answer 32

Tumour Invasion

Question 33

What types of components of signal transduction pathways are proto-oncogenes

Answer 33

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 34

What are the counter-part oncogenes of the proto-oncogenes c-Raf and c-Jun?

Answer 34

v-Raf

v-Jun

Question 35

What must be achieved for carcinoma cells to be able to metastasise?

Answer 35

• Cell-cell adhesion must be down-regulated (e.g. reduced cadherin levels)
• Cells must be motile
• Degradation of ECM must take place (matrix metalloproteinases levels increase in order to migrate through the basal lamina and interstitial ECM)

NOTE: the degree of carcinoma cell-cell adhesion is an indicator of how differentiated the primary tumour is and indicates its invasiveness and prognosis