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 by a thin wide lamellipod 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 in the 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

What is the term given to the requirement of ECM binding for growth?

Answer 5

Anchorage dependence Cells need to be attached to ECM and they need a certain degree of spreading to begin protein synthesis and proliferation (DNA synthesis) attachment to ECM may be required for cell survival

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)) peptide sequences such as RGD are found in more than one ECM molecule meaning integrins can bind more than one ECM molecule

Question 9

What do most integrins bind to intracellularly?

Answer 9

Actin cytoskeleton

Question 10

What is an exception to what most integrins bind to intracellularly?

Answer 10

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

Question 11

What do integrins form when they cluster?

Answer 11

Most integrins – focal adhesions alpha6beta4 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 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)

Question 13

Describe inside-out signalling of integrins.

Answer 13

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) The integrin goes from a low affinity ‘flexed’ position to a high affinity ‘extended’ position

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, and what is the real reason?

Answer 16

Believed reason = Contact inhibition of cell division Real reason = Density-dependence – increased competition for growth factors

Question 17

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

Answer 17

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

Question 18

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

Answer 18

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 19

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

Answer 19

Epithelial cells Endothelial cells Neurones Myocardium

Question 20

What are the two types of cell-cell junction?

Answer 20

Zonulae (belts) Maculae (spots)

Question 21

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

Answer 21

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 22

What effect does low calcium levels have on an epithelium?

Answer 22

Adherens junction is calcium dependent. Adherens jucntion is the master junction. 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 23

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

Answer 23

The same results were achieved i.e. blocking adherens junctions increased proliferation This showed that cell-adhesion affects proliferation

Question 24

Describe the structure of an adherens junction.

Answer 24

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 25

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

Answer 25

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 26

Explain how the APC mutation causes adenomatous polyposis coli.

Answer 26

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 27

What is contact inhibition of proliferation?

Answer 27

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 28

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

Answer 28

• 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 influence proliferation
• Some growth factor receptors can become incorporated into Cell-cell junctions reducing their capacity to promote proliferation

Question 29

Describe ways in which cells can lose their social skills.

Answer 29

• 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 30

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

Answer 30

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 31

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

Answer 31

Ras = V12Ras (Gly12Val mutation), L61Ras (Gln61Leu mutation) c-RAF = v-Raf c-Jun = v-Jun

Question 32

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

Answer 32

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

Question 33

What is the significance of integrins?

Answer 33

A cell can receive information about its surroundings from its adhesion to ECM The matrix type has profound effects on the phenotype of cells. mechanical continuity between ECM and the cell interior

Question 34

What signals control proliferation of tissue cells?

Answer 34

• Growth factor activation of the ERK MAP kinase cascade
• Integrin activation of the same cascade
• individually, this activation is weak and/or transient
• together, activation is strong and sustained the separate signalling pathways act synergistically