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 do integrins form when they cluster?

Answer 10

Most integrins – local adhesions

some (eg: alpha6beta4) integrin - hemidesmosomes

Question 11

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

Answer 11

It is a platform for signal transduction (outside-in or inside-out)

Question 12

Describe inside-out signalling of integrins.

Answer 12

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 13

Describe outside-in signalling of integrins.

Answer 13

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 14

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

Answer 14

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 15

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

Answer 15

Contact inhibition of cell division

Density-dependence – increased competition for growth factors

Question 16

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

Answer 16

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

Question 17

What happens to most NON-EPITHELIAL cells when they make contact with each other?

Answer 17

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 18

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

Answer 18

Epithelial cells
Endothelial cells
Neurones

Question 19

What are the two types of cell-cell junction?

Answer 19

Zonulae (belts)

Maculae (spots)

Question 20

What happens to EPITHELIAL cells when they come into contact with one another?

Answer 20

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 21

What effect does low calcium levels have on an epithelium?

Answer 21

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 22

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

Answer 22

The same results were achieved as calcium- presence of Ab blocking adherens Junction cause increased proliferation and vice versa
This showed that cell-adhesion affects proliferation

Question 23

Describe the structure of an adherens junction.

Answer 23

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 24

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

Answer 24

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 25

Explain how the APC mutation causes adenomatous polyposis coli.

Answer 25

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 26

What is contact inhibition of proliferation?

Answer 26

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 27

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

Answer 27

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 28

Describe ways in which cells can lose their social skills.

Answer 28

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- can divide indefinitly)

Question 29

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

Answer 29

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 30

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

Answer 30

v-Raf

v-Jun

Question 31

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

Answer 31

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