Mechanisms of Disease I: Cell growth and cell differentiation

Question 1

What are the basic mechanisms responsible for turning a zygote into a mature multicellular organism?

Answer 1

Cell growth = a bigger organism more cells
Differentiation = cells become complex (usually) an end to growth
Cell growth precedes differentiation, but with some overlap

Question 2

What are the two main forms of cell growth?

Answer 2

1.Hypertrophy (bigger cells)
Hypertrophy is simply cells growing bigger
More proteins, more membrane etc etc.
Elevated protein synthesis is a big driver of increased cell size

2.Hyperplasia (more cells) (more common)
more cells – is caused by cell division, or proliferation
i.e. cell cycle

Question 3

What is common between Cell growth and Differentiation?

Answer 3

The mechanisms governing them is common between cell growth and differentiation.
Cell growth and differentiation are governed by the integration of multiple signals:
intra- and extracellular signals (checks on cellular physiology, growth and inhibitory factors, cell adhesion etc.)
Signals converge on the promoters of key genes
Promoters act as “co-incidence detectors”
Express gene YES/NO? How much?

Question 4

Summarise the cell cycle

Answer 4

https://canvas.sgul.ac.uk/courses/2414/pages/mechanisms-of-disease-i-cell-growth-and-cell-differentiation-sdl?module_item_id=90323
part 2

M, G1, S, G2 are the stages overall

Question 5

What are quiescent cells?

Answer 5

Cells that have left the cell cycle (they are in G0), they reach terminal differentiation, leads eventually to apoptosis, cell death.

Question 6

What can be used to measure DNA content of cells in a population?

Answer 6

FACs
FACS (fluorescence activated cell sorting) differs from conventional flow cytometry in that it allows for the physical separation, and subsequent collection, of single cells or cell populations.
DNA stain is applied and from this, DNA content can be measured.

Question 7

Summarise Mitosis

Answer 7

Prophase (1)
Nucleus becomes less definite
Microtubular spindle apparatus assembles
Centrioles migrate to poles
Prometaphase
Nuclear membrane breaks down
Kinetochores attach to spindle in nuclear region 

Metaphase (2)
Chromosomes align in equatorial plane

Anaphase (3)
Chromatids separate and migrate to opposite poles

Telophase (4)
Daughter nuclei form
Cytokinesis
Division of cytoplasm
Chromosomes decondense

Question 8

What does the main checkpoint/restriction point (between G1 and S) check for?

Answer 8

The restriction point checks that DNA is not damaged, Cell size,
metabolite/nutrient stores

Question 9

What does the checkpoint between G2 and M phase check for?

Answer 9

Checks again that DNA is not damaged and also that DNA has completely replicated from S phase.

Question 10

What does the final checkpoint in Mitosis (M phase) check for?

Answer 10

Checks that the chromosome has aligned on the spindle

Question 11

Discuss CDK/Cyclin etc

Answer 11

CDK- Cyclin Dependent Kinase
a catalytic subunit which needs cyclin (a regulatory subunit) to function,

CDK binds with a cyclin and forms an active cyclin-CDK complex which phosphorylates specific substrate proteins

Question 12

How is the Cyclin-CDK cycle regulated

Answer 12

cycles of synthesis regulated (gene expression) and destruction (by proteasome)

Post translational modification is regulated by phosphorylation
This may result in activation, inhibition or destruction

Dephosphorylation

Also regulated by the binding of cyclin-dependent kinase inhibitors (CDKIs) (which bind to active CDK-cyclin complexes and inhibit them

Question 13

What is an example of a CDK-cyclin substrate?

Answer 13

Retinoblastoma protein (RB)
RB is a key substrate ofG1 and G1/S cyclin-dependent kinases

Normally, RB is bound to a transcription factor called E2F
Unphosphorylated RB binds E2F transcription factor preventing its stimulation of S-phase protein expression

However, in the presence of Cyclin D-CDK4
& Cyclin E-CDK2, RB becomes phosphorylated and dissociates from E2F(which E2F is then no longer supressed and therefore able to bind to the promoter of its target genes)
Released E2F stimulates expression of more Cyclin E and S-phase proteins e.g. DNA polymerase, thymidine kinase, PCNA etc.
DNA replication starts.
E2F creates a positive feedback loop

G1 CDKs are activated in response to environmental signals, late CDKs by preceding kinase activities.
Hyperphosphorylated RB is dephosphorylated by protein phosphatase 1. G1 CDKs hypophosphorylate, and late CDKs hyperphosphorylate.

Question 14

Summarise the sequence of events triggered by growth factors

Answer 14

Growth factor signalling activates early gene expression (transcription factors – FOS, JUN, MYC)

Early gene products stimulate delayed gene expression (includes Cyclin D, CDK2/4 and E2F transcription factors)

E2F sequestered by binding to unphosphorylated retinoblastoma protein (RB)

G1 cyclin-CDK complexes hypophosphorylate RB and then G1/S cyclin-CDK complexes hyperphosphorylate RB releasing E2F

E2F stimulates expression of more Cyclin E and S-phase proteins (e.g. DNA polymerase, thymidine kinase, Proliferating Cell Nuclear Antigen etc.)

S-phase cyclin-CDK and G2/M cyclin-CDK complexes build up in inactive forms. These switches are activated by post-translational modification or removal of inhibitors, driving the cell through S-phase and mitosis.

Question 15

What does DNA damage trigger?

Answer 15

3 potential:

1. Stop the cycle
2. Attempt to repair the DNA through various mechanisms
3. If repair is impossible, then programmed cell death (apoptosis)

Question 16

What is the role of TP53 (tumour protein 53)?

Answer 16

known as a ‘guardian of the genome’, it is a tumour suppressor gene

Usually, when TP53 is intact, it is continually destroyed by proteasome.
However, in response to DNA damage, this leads to kinase activation which leads to phosphorylation of P53 so it can no longer be destroyed. TP53 levels then accumulate and has effects eg stimulates expression of CDKIs (CDK inhibitors) where it will cause cell cycle arrest. Accumulation of phosphorylated TP53 also leads to activation of DNA repair mechanisms and so DNA damage can hopefully be repaired!
However, if DNA damage is not repairable, apoptosis is activated by the phosphorylated PT53.

Question 17

What can TP53 loss-of-function mutations lead to and where are they most frequent?

Answer 17

TP53 loss-of-function mutations are amongst the most frequent in cancer
Prevent cell cycle arrest-Faster growth

Prevent apoptosis-do not die

Prevent DNA repair-More mutations = more heterogeneity
= more adaptation
= cancer progression

Question 18

What is the objective of traditional chemotherapeutic drugs?

What do S phase drugs cause? (Give 2 examples)

Answer 18

Traditional chemotherapeutic drugs act on the cell cycle
Objective: stop proliferation, induce apoptosis

S-phase drugs cause DNA damage, e.g.
5-fluorouracil (prevents synthesis of thymidine)
Cisplatin (binds to DNA causing damage and blocking repair)

M-Phase drugs target the mitotic spindle
Vinca alkaloids
stabilize free tubulin
prevent microtubule polymerization
arrest cells in mitosis
Paclitaxel (Taxol)
stabilizes microtubules
preventing de-polymerization
arrests cell in mitosis
Not just cancer: colchicine (similar mode of action to vinca alkaloids) is used for immune-suppression

Question 19

Summarise key steps/stages in DNA replication/ involvement of TP3/cyclin-CDK complexes etc

Answer 19

Growth factors binding to receptors induce gene expression
G1 and G1/S Cyclin-CDK complexes phosphorylate RB in the absence of inhibition by CKIs (expression of these is regulated by TP53 or TGF)
E2F released, stimulating expression of genes required for S-phase
Cell replicates DNA (expression of S-phase Cyclin-CDK complexes)
If all DNA replicated, G2/M Cyclin-CDK complexes cause cell to enter mitosis. If chromosomes aligned on spindle, exit from mitosis is triggered
If process fails, TP53 initiates apoptosis