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

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?

Question 18

What is the objective of traditional chemotherapeutic drugs?

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

Question 19

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

Question 20

Define hyperplasia

Answer 20

increase in cell numbers (hyperplasia (most common) – cell division

Question 21

Define hypertrophy

Answer 21

increase in cell size (hypertrophy – more proteins, more membranes, elevated levels of protein synthesis. Such as the heart)

Question 22

What do hyperplasia and hypertrophy depend on?

Answer 22

Depends on the integration of intra- and extracellular signals (checks on cellular physiology, growth and inhibitory factors, cell adhesion etc.)

Question 23

What is differentiation?

Answer 23

Differentiation: exit from the cell cycle, known as post-mitotic cells. It involves a program of cell type-specific gene expression. The cell morphology and function changes.

Question 24

What is programmed cell death called?

Answer 24

• Loss of cells by programmed cell death (apoptosis)
o A coordinated program of cell dismantling ending in phagocytosis. Distinct from necrosis

o Occurs during normal development (e.g. separation of the digits, involution, immune and nervous system development)
o And in response to DNA damage and viral infection

Question 25

Is there anything in common between cell growth and differentiation?

Answer 25

• Yes – the mechanisms governing them
• Cell growth and differentiation are both governed by the integration of multiple signals:
• Depends on integration of intra- and extracellular signals (checks on cellular physiology, growth and inhibitory factors, cell adhesion etc.)
• These signals converge on the promoters of key genes – these promoters act as “co-incidence detectors.” These then determine if the gene is expressed and how much expression.

Question 26

What are Growth factors, cytokines and interleukins?

Answer 26

Proteins that:
• Stimulate proliferation (called mitogens) and maintain survival

o Usually named after originally identified target e.g. EGF, FGF, Interleukins (IL2 & IL4), NGF
o But see also PDGF (platelet-derived GF) and IGF1 (Insulin-like GF – the main effector of pituitary growth hormone)
• Stimulate differentiation and inhibit proliferation e.g. TGF Beta (transforming growth factor)
• Induce apoptosis e.g. TNFα and other members of the TNF family (tumour necrosis factor)

Question 27

briefly explain how extracellular signals work

What are the three broad classes of extracellular

Answer 27

Ligand – receptor – intracellular cascade
Three broad classes:

1. Paracrine: produced locally to stimulate proliferation of a different cell type that has the appropriate cell surface receptor
2. Autocrine: produced by a cell that also expresses the appropriate cell surface receptor
3. Endocrine: like conventional hormones, released systemically for distant effects

Question 28

Describe cell population growth

Answer 28

• When you add growth factor, the cells will respond and enter the cell cycle and start to divide
• If the PDGF is no longer available, there is a plateau until they receive more PDGF

o If the cells receive TGF Beta, they will stop dividing
• TNF Alpha will lead to the cells committing suicide and the number of cells going down
• So, we can influence cell number according to what factors we add

Question 29

What are the phases of the cell cycle?

Answer 29

• M is mitosis. This is the separation of the chromosomes and the physical separation of the cell into 2 daughter cells
o After the M phase, one of the daughter cells undergoes interphase. The cell grows using nutrients to create more cytoskeleton etc.

• Synthesis is when DNA replication takes place  two copies of the genomic DNA
• In the growth phases the cells get ready, so they have everything that is required for mitosis
• Quiescent Cells are cells that are arrested in the G0 phase
o These cells can re-enter the cell cycle if we add mitogens and will start proliferating
o Some quiescent cells may start to differentiate. E.g. they could become gut cells. This is called terminal differentiation. Some cells undergo apoptosis and die
• After cell division, the cells have two copies of each chromosome so that is 2N
o After the S phase, there is more duplication, so it is 4N

Question 30

What does Fluorescence activated cell sorter (FACS) analysis. show us?

Answer 30

• In G1 phase 2N- diploid state, in M phase 4N state as chromosome has been duplicated.
• Take cells and label the DNA with dye.

• The dye is read by a laser and the laser tells us how intense the cells are in each phase.
o We can see in what stage cells are in.
• This graph shows the difference between a slow and fast rate of proliferation.
o In the high rate of proliferation, there are a lot more cells that are in the S phase

Question 31

Describe the principles of DNA replication

Answer 31

• DNA is replicated semiconservatively (daughter cells inherit one parental and one new strand)
• New DNA is synthesised in the 5’ to 3’ direction from deoxynucleotide triphosphate precursors at a replication fork by a multienzyme complex (a replication machine)
• Fidelity is determined by base pairing (A=T, G≡C) and presence of a proof-reading enzyme in DNA polymerase
• Synthesis of the new DNA strand uses an RNA primer and occurs continuously on the leading strand and discontinuously on the trailing strand (giving rise to Okazaki fragments, which are ligated together after removal of the RNA primer)

Question 32

What do the different stains show?

Answer 32

• Stain in blue is for DNA
• Stain in red is for gamma tubulin which is required to form the microtubules that will bind to the centrioles and chromosomes

• Stain in green is for CHEK2 which is a cell cycle checkpoint protein

Question 33

What are the phases of mitosis?

Answer 33

• Prophase (1)
• Nucleus becomes less definite

• Microtubular spindle apparatus assembles
• Centrioles (yellow) migrate to poles
• This is due to the overlap in the green and the red
• Prometaphase
o Nuclear membrane breaks down
o Kinetochores attach to spindle in nuclear region
• Metaphase (2)
o Chromosomes (blue) align in equatorial plane
• Anaphase (3)
o Chromatids separate and migrate to opposite poles
• Telophase (4)
o Daughter nuclei form
• Cytokinesis
o Division of cytoplasm
o Chromosomes decondense

Question 34

What drugs affect the S-phase of the cell cycle:
5-Fluorouracil
Bromodeoxyuridine
Tamoxifen

Answer 34

• 5-Fluorouracil (an analogue of thymidine blocks thymidylate synthesis). So, DNA can’t make 2 copies (duplicate), so arrested in s phase of cell cycle.
• Bromodeoxyuridine (another analogue that may be incorporated into DNA and detected by antibodies to identify cells that have passed through the S-phase).
• Tamoxifen is an antagonist of oestrogen, stops cell growth.
• We know that breast cells need oestrogen to work so we can use it to decrease proliferation in ER-positive breast cancer cells

Question 35

What drugs affect the M-phase of the cell cycle:
Colchicine
Vinca alkaloids
Paclitaxel

Answer 35

• Colchicine (stabilises free tubulin, preventing microtubule polymerisation and arresting cells in mitosis – used in karyotype analysis), so chromosomes cannot separate , arrest at M phase.
• Vinca alkaloids (similar action to colchicine)

• Paclitaxel (Taxol, stabilises microtubules, preventing de-polymerisation)

5-Fluorouracil, paclitaxel, the vinca alkaloids and tamoxifen are used in treatment of cancer. The idea is to stop cancer cells dividing.

Question 36

What is the purpose of checkpoints within the cell cycle?

Answer 36

Controls (involving specific protein kinases and phosphatases) ensure the strict alternation of mitosis and DNA replication.

Question 37

What are protein kinases?

Answer 37

Protein kinase – regulates by phosphates do the opposite job) add phosphate groups to substrates.

Question 38

What does Cyclin-dependent kinase activity control?

Answer 38

Cell cycle progression

o Cell cycle is controlled by CDK.
o To be active, it needs to form a complex with Cyclin.
o The complex recognises substrate proteins and is then phosphorylated.

Question 39

How is Cyclin-CDK Activity regulated?

Answer 39

• Cyclical synthesis (gene expression) and destruction (by proteasome).
• Post translational modification by phosphorylation – depending on modification site may result in activation, inhibition or destruction
• Dephosphorylation
• Binding of cyclin-dependent kinase inhibitors

Question 40

What is the retinoblastoma protein?

Answer 40

The retinoblastoma protein is a key substrate of G1 and G1/S cyclin-dependent kinases

• The RB is a key substrate of G1 and G¬1/S cyclin-dependent kinases
• RB in cells that are in g0/g1 adds TF E2F.
• E2F regulates many genes that enter s phase , e.g. DNA Pol

Question 41

What are the 2 types of Cyclin-dependent kinase inhibitors?

Answer 41

o Two families of CKIs:
1) CDK Inhibitory Protein/Kinase Inhibitory Protein (CIP/KIP) family (now called CDKN1)

– Expression of members of this family stimulated weakly by TGF and strongly by DNA damage (involving TP53)
 TP 53 Tumour Suppression gene, activates expression of kinase inhibitors
– Inhibit all other CDK-cyclin complexes (late G1, G2 and M)
– Are gradually sequestered by G1 CDKs thus allowing activation of later CDKs
2) Inhibitor of Kinase 4 family (INK4) (now called CDKN2)
– Expression stimulated by TGF
– Specifically inhibit G1 CDKs (e.g. CDK4 the kinase activated by growth factors

Question 42

How do growth factors induce cyclin expression?

Answer 42

• A growth factor binds to its receptors and activates signal transducers
• There are effects in the nucleus and different waves of transcription factor will be activated
• Cell from G0,
• Cells receive GF, receptors on cell recognise GF they bind, activate intracellular pathways, leads to affect in nucleus regulates expression of P21/Cyclin

Question 43

What is TP53 and how does it act?

Answer 43

• Intact DNA molecule has a mutation
o The damage is detected by kinases

o These kinases activate CHEK2  TP53 is a substrate for CHEK2
• P53 is expressed in cells however is the protein is not functional as it is quickly degraded by the proteasome.
• In response to DNA damage, kinases phosphorylate P53.
o When it is phosphorylated, it cannot be degraded and it now stabilised and active.
o It will now go and bind the promoters of transcription factors and will help to express genes that are required for DNA repair.
 If a cell cannot be repaired, P53 will trigger apoptosis which gets rids of cells that are deleterious for the whole organism.