External Factors

Question 1

Define cell behaviour

Answer 1

Term used to describe the way in which cells interact with their external environment and their reactions to this, particularly to
• proliferative
AND
• motile responses of the cell

Question 2

What external influences are detected by cells?

Answer 2

Chemical
• e.g. hormones, GFs, [ion], ECM etc.

Physical
• e.g. mechanical stress, oC, topography of ECM

Question 3

What are the MAIN external factors the can influence cell division in relation to cancer cell behaviour?

Answer 3

• GFs
• Cell-cell adhesion
• Cell-ECM adhesion

Question 4

Explain basic cell behaviour

Answer 4

A cell will normally spread on a culture surface and thus acquire motility and polarity
• the cell will have an obvious direction to it

This process is an energy-dependant event - required to
• modulate cell adhesion
AND
• the cytoskeleton of the cell

Question 5

What helps a cell to progress into the s-phase?

Answer 5

Cell-ECM adhesion!

If it SPREADS OUT effectively on the adhesive patch
• cells require binding to ECM to be fully competent
• the cell may apoptose if it can NOT bind enough to ECM

Question 6

Why is attachment to the ECM so important for cells?

Answer 6

In suspension, cells do NOT significantly synthesise DNA or protein, the require to be attached to ECM (and a degree of spreading) to do this

Attachment to the ECM may be required for SURVIVAL
• – Anchorage Dependency

Question 7

A cell can receive information about its surroundings from its adhesion to ECM - how do they do that?

Answer 7

1. Cells have receptors on their surface for ECM molecules
2. Receptors are linked to the cytoskeleton inside the cell
3. Leading to a mechanical continuity between the ECM and the cytoplasm

Question 8

What helps to the attach the cell cytoskeleton to the ECM by biochemically sensing whether adhesion has occurred?

Answer 8

Integrins

Question 9

Explain the structure of integrins and how the function

Answer 9

Integrins are heterodimers of:
• alpha & beta sub-units that associate at the head (extracellular) region
• ~10 alpha and 8 beta sub-units are known and >20 combinations

They recognise short, specific peptide sequences and each combination of subunits of an integrin binds to a specific sequence
 IMPORTANT – the same sequence may be found in multiple types of ECM molecule.

Integrins can also bind to specific adhesion molecules found on other cell surfaces

Question 10

How are integrins involved with the cytoskeleton?

Answer 10

Integrins are linked via ACTIN proteins to the actin cytoskeleton
• Integrins cluster to form FOCAL ADHESIONS (most) or HEMI-DESMOSOMES
- alpha6beta4-integrin is found in epithelial hemi-desmosomes

• the clusters are involved in signal transduction

Question 11

What is meant my ‘outside-in’ integrin signalling and explain this?

Answer 11

ECM receptors can act to transduce signals INSIDE THE CELL when stimulated

• A cell can receive information about its surroundings from its adhesion to the ECM which can alter the phenotype of the cell
 E.G. In interstitial matrix (T1 collagen), mammary epithelium does not differentiate to secretory cells but in basal lamina ECM, it does organise and differentiate.

• The amount of force generated at a focal adhesion depends on both:
 The force generated by the cytoskeleton (F cell).
 The stiffness of the ECM.

Question 12

What is meant by ‘inside-out’ integrin signalling and explain this

Answer 12

Signals generated INSIDE THE CELL can act on the integrin to alter the affinity of it
• e.g. as the result of a hormone binding to receptor

• I.E. in inflammation or blood-clotting, this switches on adhesion of circulating leukocytes
• The inside signals flex the receptor outwards so it has more affinity for the ligands binding.
• Integrins recruit cytoplasmic proteins which can promote signalling and actin assembly.

Question 13

What is meant by Density-dependence of cell division and what opposes this theory?

Answer 13

i.e. Contact Inhibition

At high density, cells compete for GFs
• when cells form a confluent monolayer, they stop proliferating because the GFs have been all used up

This is a competing theory for contact inhibition
• that when cells meet each other in a mono-layer, they stop proliferating due to the contact of the cells.

Question 14

What helps control proliferation?

Answer 14

There is cross-talk between the
• ECM (anchorage-dependency)
AND
• the growth factor signalling (density dependency)

which leads to the proliferation of the cell.

Question 15

Mechanism of anchorage dependence?

Answer 15

GF receptors AND integrin signalling can each activate identical signalling pathways (e.g. MAPK, MAPKK, etc.)

Synergistic relationship – individually, the activation is weak but when BOTH GF and ECM anchorage activate the pathways, the activation is strong and sustained

Question 16

What are the 2 types of contact interactions between cells?

Answer 16

SHORT-TERM
• transient interactions between cells which do NOT form stable cell-cell junctions

LONG-TERM
• stable interactions resulting in formation of cell-cell junctions

Question 17

Explain the cell-cell contact between non-epithelial cells

Answer 17

SHORT-TERM!

When most non-epithelial cells collide, they do NOT form cell-cell contacts
• they repel each other by paralysing motility at the contact site
AND
• promoting formation of a motile front at another site

This is called CONTACT INHIBITION OF LOCOMOTION
• prevents multi-layering of cells

Question 18

Explain the cell-cell contact between epithelial, endothelial and neuronal cells

Answer 18

LONG-TERM

These cells adhere strongly to each other and form cell-cell junctions
• i.e. adherens
• i.e. desmosomes
• i.e. tight junctions (gap junctions)

Epithelial & endothelial cells = form layers
Neurones = form synapses

Question 19

Explain how cell-cell junctions in epithelia are arranged and the link with proliferation

Answer 19

Junctions are usually arranged as
• continuous belts (zonula)
or
• discrete spots (Macula)

Cell-cell adhesion
• +Ca2+ & LESS adhesion-blocking-antibody:
• –> cell-cell junctions, inactive MAPK, increased p27KIP1 and low proliferation
• vice versa = high proliferation

Question 20

What are Cadherins

Answer 20

Ca2+-dependent, homophillic cell adhesion molecules

Question 21

Which Cadherin is thought to be a possible link to cell-cell adhesion?

Answer 21

B-catenin

• In APC (Adenomatous Polyposis Coli), the APC gene-product is a protein involved in the breakdown of the B-catenin molecule

APC - inherited colon cancer

Question 22

Explain how B-catenin functions depending on where it is

Answer 22

In its normal state:
• B-catenin is bound to cadherin in the membrane BUT it can pass to the cytoplasm

From the cytoplasm, B-catenin can:
• Rapidly degrade when bound to APC
• When at a high enough concentration, can bind to LEF-1 in the nucleus and alter gene transcription = PROLIFERATION of cells
• Re-bind back onto cadherin (cannot have nuclear effects)

Question 23

In relation to B-catenin, how can it help in the proliferation of cells?

Answer 23

If B-catenin levels rise because of
• inhibition of degradation (FAP/APC)
or
• loss of cadherin-mediated adhesion,

B-catenin/LEF-1 complexes can then enter the nucleus and influence gene expression –> proliferation of cells.

Question 24

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

Answer 24

Clustering of cadherins (after cell-cell contact) alters the 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 25

Under certain condition, cells lose their behavioural constraints - what are they?

Answer 25

o Proliferate uncontrollably – no density-dependency

o Less adherent, will multilayer – lose contact-inhibition and anchorage dependency

o Epithelial breakdown of cell-cell contacts

o Not Hayflick limited so express telomerase

Question 26

What does loss of contact inhibition mean in cancer cells?

Answer 26

PROMOTE
• formation of solid tumours
AND
• local invasion

Question 27

If the gene encoding signalling pathway is mutated, what does that mean?

Answer 27

In CANCER!

If the gene encoding signalling pathways is mutated, the protein may be constitutively active so the pathway is ALWAYS ON
• This is the mechanism of short-circuiting –> uncontrolled proliferation because of loss of GF dependency

(Ludley picture!)

Question 28

Oncogenes vs. Proto-oncogenes?

Answer 28

Oncogenes
• mutant genes which promote uncontrolled proliferation

Proto-oncogenes
• normal genes corresponding to the oncogene
e.g. receptors, signalling intermediates/targets

Question 29

What is mutated in ~30% of ALL cancers?

Answer 29

Ras

Question 30

In addition to deregulated proliferation, what else is a major feature of cancerous tumours?

Answer 30

Ability to SPREAD

Question 31

How does this other major feature of cancerous tumour arise?

Answer 31

Most human cancers are carcinomas (of epithelial origin)
• to spread, the cells must break away and form a secondary tumour, distant from the primary

Mechanism of metastasis:
o Cell-cell adhesion broken down-regulated
o Cells must be motile
o Degradation of ECM (via MMPs) to migrate

The degree of cell-cell adhesion is an indicator of how differentiated the primary tumour is and indicates its invasiveness and the prognosis.