External factors controlling division and behaviour of normal and cancerous cells

Question 1

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

Answer 1

Growth factor
Cell-cell adhesion
ECM-cell adhesion

Question 2

Describe what happens to a cell when it is placed on a culture medium

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, it is not just happening because of gravity. Energy is required to modulate cell adhesion and changes inthe cytoskeleton during spreading

Question 3

Describe what happens to cells placed on:

a. Non-adhesive agar
b. Small adhesive patch
c. Large adhesive patch

Answer 3

a. Non-adhesive agar
Very few cells enter S phase
b. Small adhesive patch
A small proportion of cells will proliferation
c. Large adhesive patch
Almost all the cells will start proliferating

Question 4

What is the difference in proliferation when a cell is placed on:

a. A small patch of fibronectin
b. The same amount of fibronectin spread over a larger area

Answer 4

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 5

Cells need to be attached to ECM and they need a certain degree of spreading to be able to respond to soluble growth factors. What is the term given to the requirement of ECM binding for growth?

Answer 5

Anchorage dependence

Question 6

Describe the structure of integrins.

Answer 6

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

Question 7

How many different alpha and beta subunits are there?

Answer 7

10 alpha and 8 beta

There are over 20 known combinations

Question 8

What do the extracellular parts of integrins bind to?

Answer 8

Short, specific peptide sequences (e.g. arg-gly-asp (RGD sequence))

Question 9

What do most integrins bind to intracellularly?

Answer 9

Actin cytoskeleton

Question 10

What is an exception to this generalisation?

Answer 10

The alpha6beta4 integrin is found in hemidesmosomes in epithelia and it binds to cytokeratin instead

Question 11

What do integrins form when they cluster?

Answer 11

Most integrins – local adhesions

al[ha6beta4 integrin - hemidesmosomes

Question 12

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

Answer 12

It is a platform for signal transduction

Question 13

Describe inside-out signalling of integrins.

Answer 13

Growth factors can generate signals inside the cell, which can act on the integrin complex and alter its affinity (this is important in bloodclotting)

Question 14

Describe outside-in signalling of integrins.

Answer 14

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 15

Describe how the experiment with cultures of mammary epithelium demonstrated the profound effect of ECM on the phenotype of cells.

Answer 15

When the mammary cells were placed on a culture medium with interstitial matrix (type 1 collagen), they formed clumps and were loosely associated
When placed on a culture medium with basement membrane matrix (e.g. laminin), the cells formed a very ordered system (organoid) and even began producing milk proteins

Question 16

When cells are dividing on a culture medium, they will stop dividing once they reach the edges of the medium. What was originally thought to be the reason behind this?

Answer 16

Contact inhibition of cell division

Question 17

What is the actual reason for this?

Answer 17

Density-dependence – increased competition for growth factors

Question 18

In summary, what two pathways work synergistically to trigger proliferation in cells?

Answer 18

Anchorage dependence (ECM dependent) 
Density dependence (growth factor dependent)

Question 19

What happens to most non-epithelial cells when they make contact with each other?

Answer 19

They will move away from each other
The motility on the side that made contact will become paralysed meaning that the cells can then move away from each other
This prevents multi-layering
This is CONTACT INHIBITION OF LOCOMOTION

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?

Answer 22

Contact-induced spreading of epithelial cells
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

What effect does low calcium levels have on an epithelium?

Answer 23

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 24

What effects do antibodies blocking adherens junctions have on an epithelium?

Answer 24

The same results were achieved

This showed that cell-adhesion affects proliferation

Question 25

Describe the structure of an adherens junction.

Answer 25

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 beta-catenin, which is bound to alpha-catenin, which, in turn, is bound to the actin cytoskeleton

Question 26

Describe the action of beta-catenin when it isn’t sequestered by cadherin at the plasma membrane.

Answer 26

Normally, beta-catenin is rapidly degraded by APC so it doesn’t tend to achieve high concentrations in the cytoplasm
Free beta-catenin in the cytoplasm (that is not broken down by APC) can bind to LEF-1 to form a complex that acts as a transcription factor
This complex can then regulate gene expression and promote proliferation

Question 27

Explain how the APC mutation causes adenomatous polyposis coli.

Answer 27

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

Question 28

What is contact inhibition of proliferation?

Answer 28

When bound to cadherin at the plasma membrane, beta-catenin is NOT available to bind to LEF-1 and cause nuclear effects
Cytoplasmic levels of beta-catenin can rise if there is:
 Inhibition of degradation
 Loss of cadherin-mediated adhesion
This can lead to the formation of beta-catenin/LEF-1 complexes that promote proliferation

Question 29

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

Answer 29

When cadherins cluster together you can get changes in the activation of some of the small GTPases including Rac and Rho
Changes in the small GTPases can induce proliferation

Question 30

Describe ways in which cells can lose their social skills.

Answer 30

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 31

What types of components of cell proliferation tend to be oncogenes?

Answer 31

Anything that will make the signalling pathway constitutively active e.g. Ras mutation, over-expression of growth factor receptors, signalling intermediates and signalling targets

Question 32

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

Answer 32

v-Raf

v-Jun

Question 33

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

Answer 33

Cell-cell adhesion must be down-regulated (e.g. reduced cadherin levels)
Cells must be motile
Degradation of ECM must take place (matrix metalloproteinases (MMP) levels increased 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