External Factors Controlling Division and Behaviour of Normal and Cancerous Cells

Question 1

What is cell behaviour?

Answer 1

the way cells interact with their external environment and their reactions to this, particularly proliferative and motile responses of cells

Question 2

What external influences to cells detect?

Answer 2

Chemical influences
- Hormones
- growth factors
- ion concentrations
- ECM (density, composition)
- molecules on other cells
 nutrients and dissolved gas (O2/CO2) concentrations

Physical influences

• mechanical stresses
• temperature
• the topography or “layout” of the ECM and other cells (the organisation of the ECM

Question 3

What external factors can influence cell division?

Answer 3

• Growth factors
• Cell-cell adhesion
• Cell-ECM adhesion

Question 4

How do tissue cells acquire motility on a culture surface?

Answer 4

Cells will initially adhere to a culture surface, and when the culture is turned upside down, the cells will STILL spread out. This process requires energy, adhesion and pushing out of cellular margins to EXPAND.

the cell initially adheres, before flattening and sending out lamellipods and filopodia

Question 5

When do growth factors work on the cells in a culture?

Answer 5

only when its fully spread out

Question 6

How does the way adhesion islands are distributed affect the spread of cells in a culture?

Answer 6

e.g fibronectin

When fibronectin
(a matrix molecule that sticks to culture surfaces) is a single spot, the cell can adhere but it cannot spread very far. On a larger area (same amount of fibronectin, better distribution), it can spread, survive and proliferate.

Question 7

What allows for mechanical continuity between ECM and the cell interior?

Answer 7

arrangement of cell surface receptors on cell surface

Question 8

What are integrins?

Answer 8

• Integrins are heterodimer complexes of alpha and beta subunits
• They associate extracellularly by their “head” region
• The beta subunit has a slightly longer cytoplasmic tail
• there are more than 20 different combinations known
• Integrins recognise short, specific peptide sequences within matrix molecules
• Each combination specifically binds a particular peptide sequence - can be found in more than one ECM molecule

Question 9

How do integrins link?

Answer 9

Most integrins are linked, via actin-binding proteins, to the actin cytoskeleton (indirect association)

• a6b4 integrin complex found in epithelial hemidesmosomes, linked to the cytokeratin (intermediate filament) network is the ONLY integrin that we know of that ISN’T associated with the actin cytoskeleton

Question 10

What do integrins cluster to form?

Answer 10

focal adhesions (most) or hemidesmosomes (a6b4) - normally found at the ends of bundles of filamentous actin (the microfilaments of the cytoskeleton)

• clusters are involved in signal transduction
• Integrins also bind to specific adhesion molecules on some OTHER cells
• E.g. avb3 binds to PECAM-1 (CD31)
• E.g. aIIbb2 to ICAM-1 on endothelial cells in inflammation

Question 11

What is outside-in integrin signalling?

Answer 11

A molecule OUTSIDE the cell stimulates a signal INSIDE THE CELL e.g ECM binding to an integrin complex can stimulate the complex to produce a signal inside the cell

• composition of the ECM will determine which integrin complexes bind and which signals it receives
• This can alter the phenotype of the cell

Question 12

What is the role of focal adhesions?

Answer 12

sense the mechanical properties of their surroundings - cells can detect the amount of force in their environment

The amount of force that is generated at a focal adhesion depends on both the force generated by the cytoskeleton (F cell) and the stiffness of the ECM

Question 13

How do integrins promote signalling?

Answer 13

The alpha and beta heterodimers DO NOT have ANY enzymatic activity in themselves. So they CANNOT signal directly - they CAN recruit other molecules, some of which are SIGNALLING MOLECULES WHICH ASSOCIATE WITH THE ACTIN CYTOSKELETON.

Question 14

What happens to mammary epithelium cells when you culture them on type 1 collagen or basal lamina matrix gel?

Answer 14

TYPE 1 COLLAGEN:
Cells cluster together, but the clusters are quite loose and undifferentiated

BASAL LAMINA MATRIX GEL:
Cells form a spherical cyst (hollow ball) and they switch on the production of milk proteins - there were hormones present (required to produce milk in these cells)

Question 15

What happens to mammary epithelium cells when you culture them on type 1 collagen or basal lamina matrix gel?

Answer 15

TYPE 1 COLLAGEN:
Cells cluster together, but the clusters are quite loose and undifferentiated

BASAL LAMINA MATRIX GEL:
Cells form a spherical cyst (hollow ball) and they switch on the production of milk proteins - there were hormones present (required to produce milk in these cells)

Question 16

What does integrin activation involve?

Answer 16

significant conformational change

• The low affinity, ‘bent’ conformation of the integrin doesn’t bind well to matrix proteins.
• An intracellular signal can influence the conformation of the integrin, pushing it to the extended high affinity conformation, which is capable of binding the ligand.
• Ligand binding causes a further change to the molecule, to allow cytoplasmic signalling molecules and linkers to bind

Question 17

What is density dependence of cell division?

Answer 17

at high density, there is too much competition for groth factors - proliferation stops, metabolic activities slow

*used to be know as contact inhibition because they stopped proliferating when they made a confluent monolayer on the culture

Question 18

What happens in the ERK MAP kinase cascade?

Answer 18

Adaptor molecules recruit Ras protein, which activates RAF -> MEK -> ERK (these are various levels of MAP-kinases)

• can influence gene expression, and stimulate proliferation. However, we know that this is not enough to stimulate proliferation on its own, because cells need to be bound to matrix (anchorage dependence)

Question 19

What is the mechanism of anchorage dependence?

Answer 19

GF receptors and integrin signalling complexes can activate identical signalling pathways (e.g. MAPK)

Individually, this activation is weak and/or transient but together, activation is strong and sustained

The separate signalling pathways act synergistically

Question 20

What are the contact interactions between cells?

Answer 20

Short-term: transient interactions between cells which do not form stable cell-cell junctions

Long term: stable interactions resulting in formation of cell-cell junction

Question 21

What is contact inhibition of locomotion?

Answer 21

when most non-epithelial cells “collide”, they do not form stable cell-cell contacts - they actually “repel” one another by paralysing motility at the contact site
- promotes the formation of a motile front at another site, and moving off in the opposite direction

• responsible for preventing multi-layering of cells in culture and in vivo

Question 22

What happens in contact induced spreading of epithelial cells?

Answer 22

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

Question 23

How does calcium affect cell adhesion?

Answer 23

junctions are Ca dependent - won’t form unless there’s calcium

When there are NO cell-cell junctions, MAPK is activated, p27KIP1 is decreased -> HIGH PROLIFERATION

If calcium is added, junctions re-form -> inactive MAPK, p27KIP1 is increased -> LOW PROLIFERATION

• antibodies would bind to cell adhesion molecules and BLOCK ADHESION (same as absent ca)

Question 24

What does beta-catenin do?

Answer 24

• links the cell junction molecule to the actin cytoskeleton (structural).
• acts as signalling molecules
• when it is in the cytoplasm, it can bind with LEF-1, enter the nucleus and alter cell proliferation (it acts like a transcription factor)

Question 25

What does the APC gene product do?

Answer 25

• Adenomatous polyposis coli

a protein involved in the degradation of the junction-associated molecule, beta-catenin

If the APC gene is faulty, the degradation is reduced and there is an increase in cytoplasmic beta-catenin, which will associate with LEF-1. This leads to increased cell proliferation

Question 26

What other adhesion-associated signalling pathways influence contact-induced inhibition of proliferation?

Answer 26

• 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 - reduces their capacity to promote proliferation

Question 27

What happens when cells lose their behavioural restraints?

Answer 27

• Proliferate uncontrollably (lose density dependence of proliferation)
• Are less adherent and will multilayer (lose contact inhibition of locomotion and anchorage dependence)
• Epithelia breakdown cell-cell contacts
• Not Hayflick limited, express telomerase i.e. cancer

Question 28

How does loss of growth factor dependence lead to uncontrolled proliferation?

Answer 28

Gene coding for a component of a signalling pathway may be mutated so that the protein is active so that pathway will be permanently ‘on’

Receptors, signalling intermediates and signalling targets (e.g. TFs) are proto-oncogenes

may not need GFs or integrin complex signalling. Therefore, the density dependence and anchorage dependence is lost. This has consequences for things like SPREADING

Question 29

What is a local invasion/ metastasis?

Answer 29

Where cells break away from the original tumour and begin to invade

to spread - travel to a blood or lymph vessel, enter the vessel, lodge at a distant site, leave the vessel, and ultimately establish a secondary tumour

Question 30

How does a primary carcinoma cell metastisise?

Answer 30

• Cell-cell adhesion must be down-regulated (e.g. cadherin levels reduced)
• cells must be motile
• Degradation of ECM must take place; matrix metalloproteinase (MMP) levels increased in order to migrate through basal lamina and interstitial ECM