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
Cell-ECM adhesion

Question 2

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

Answer 2

Settles + spread across the surface
Gains some sort of polarity
Becomes motile
= Active process, not just happening because of gravity. Energy required to modulate cell adhesion + changes in 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

Non-adhesive agar: Few cells enter S phase
Small adhesive patch: Small % of cells proliferate
Large adhesive patch: Almost all cells 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

Small patch of fibronectin: cell can stick but can’t spread so dies via apoptosis
Same amount of fibronectin spread over a larger area:
Cell is able to stick + spread so survives + grows
This shows adhesion + spreading is important for cell survival + proliferation

Question 5

Cells need to be attached to ECM and 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

Heterodimer complexes of alpha + beta subunits
Associate extracellularly via their “head”
Each of the “leg” regions spans the plasma membrane

Question 7

How many different alpha and beta subunits are there?

Answer 7

10 alpha + 8 beta

> 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

Which integrin does not bind to the actin cytoskeleton intracellularly? What does it bind to?

Answer 10

Alpha6Beta4 in hemidesmosomes in epithelia

Binds to cytokeratin

Question 11

What do integrins form when they cluster?

Answer 11

Most integrins: Focal adhesions

Alpha6Beta4: Hemidesmosomes

Question 12

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

Answer 12

Involved in signal transduction

Some bind to specific adhesion molecules on other cells e.g. in inflammation

Question 13

Describe inside-out signalling of integrins.

Answer 13

A signal generated inside the cell can act on an intergrin complex + alter its affinity for ECM binding (important in blood clotting)

Question 14

Describe outside-in signalling of integrins.

Answer 14

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

On a culture medium with interstitial matrix (type 1 collagen) formed loose clumps + stayed undifferentiated
On a culture medium with basement membrane matrix (e.g. laminin), the cells formed a very ordered system (organoid) + began producing milk proteins

Question 16

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

Answer 16

Contact inhibition of cell division

Question 17

What is the actual reason cells stopping dividing on a culture medium once they reach the edges?

Answer 17

Density-dependence: increased competition for external growth factors

Question 18

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 don’t form stable cell-cell contacts
“Repel” each other; motility on the side of contact will be paralysed promoting formation of a motile front at another site on the cell
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 (form layers)
Endothelial cells (form layers)
Neurones (form synapses)

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 the total spread area of the contacted cells is greater than the sum of the 2 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, junctions break down
This leads to:
Increased MAPK activation
Decreased activity of p27KIP1 (Cdk inhibitor)
INCREASED PROLIFERATION
When calcium returned to normal + junctions 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

Increased MAPK activation
Decreased activity of p27KIP1 (Cdk inhibitor)
INCREASED PROLIFERATION
This showed that cell-adhesion affects proliferation

Question 25

Describe the structure of an adherens junction.

Answer 25

Transmembrane Cadherin domain that projects extracellularly
Cadherins are Ca2+ dependent + homophilic so associate with similar structures on adjacent cells
Intracellularly, 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 reach high concentrations in the cytoplasm
Free beta-catenin in the cytoplasm (not broken down by APC) can bind to LEF-1 to form a transcription factor complex
This enters the nucleus, influences gene expression + promotes proliferation

Question 27

Explain how the APC mutation causes adenomatous polyposis coli.

Answer 27

APC protein no longer degrades beta-catenin efficiently

So beta-catenin accumulates in the cytoplasm, associated with LEF-1 + triggers increased proliferation

Question 28

What is the mechanism of contact inhibition of proliferation?

Answer 28

When bound to cadherin at the membrane, beta-catenin is NOT available to bind to LEF-1 + 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 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

Clustering of cadherins after cell-cell contact alters activation of small GTPases: Rac is activated + Rho is inhibited. This can influence proliferation.
Some GF receptors are associated with cell-cell junctions- this reduces their capacity to promote proliferation

Question 30

Describe ways in which cells can lose their social skills.

Answer 30

Proliferate uncontrollably (lose density dependence of proliferation)
Become less adherent + multi-layer (lose contact inhibition of locomotion + anchorage dependence)
Epithelia breakdown cell-cell contacts
Not hayflick limited (express telomerase + 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 + 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)
Cells must be motile
Degradation of ECM must take place (matrix metalloproteinases (MMP) levels increased in order to migrate through basal lamina + interstitial ECM)
Degree of carcinoma cell-cell adhesion is an indicator of how differentiated primary tumour is + indicates its invasiveness + prognosis

Question 34

How does the type of matrix a cell is bound to effect it?

Answer 34

Effects the phenotype of the cell

Suggests cells can sense composition of their environment from adhesion to ECM

Question 35

What allows mechanical continuity between ECM and cell interior?

Answer 35

Cell receptors that span the membrane linked at their cytoplasmic domains to the cytoskeleton

Question 36

What are the 2 conformations integrin complexes may adopt?

Answer 36

Flexed: Legs bent, head down
Extended: Legs extended + open, head up

Question 37

What is an oncogene?

Answer 37

Mutant gene which promotes uncontrolled cell proliferation

Question 38

What is a proto-oncogene?

Answer 38

Normal cellular gene with the potential to become an oncogene

Question 39

Give 4 examples of proto-oncogenes

Answer 39

EGF receptor
Ras
c-Raf
c-Jun