Mechanisms of Disease I

Question 1

What is cell growth?

Answer 1

Increase in number of cells

Question 2

What is differentiation?

Answer 2

Specialisation of cells where they become more complex. (Usually) an end to growth.

Question 3

What are the three main groups associated with disease related cell growth and differentiation?

Answer 3

1. Developmental conditions – defects due to cell growth and/or differentiation.
2. Neoplasia (and metaplasia – transformation of one cell type into another – usually pre-cancerous growth) e.g., cancers, tumours.
3. Others e.g., cardiac hypertrophy

Question 4

What are the two main forms of cell growth?

Answer 4

Hypertrophy – bigger cells and hyperplasia – more cells

Question 5

How does hypertrophy take place?

Answer 5

Less common mechanism of cell growth in humans. It’s caused by cells making more macromolecules e.g., proteins, lipids, membranes. Elevated protein synthesis is a sufficient driver of increased cell size.

Question 6

How does hyperplasia take place?

Answer 6

It’s caused by increased cell division or proliferation via the cell cycle.

Question 7

How does differentiation take place?

Answer 7

It’s when cells exit the cell cycle. They are post-mitotic. Each cell in differentiation starts to elicit a program of cell-type specific gene expression. Cell morphology and function change e.g., stem cells to cardia myocytes to heart.

Question 8

What are cell growth and differentiation regulated by?

Answer 8

They are governed by the integration of multiple signals. Intra and extracellular signals (checks on cellular physiology (intra), growth and inhibitory factors, cell adhesion (extra) etc.)
These signals are integrated/converge on the promoters of key genes for proliferation/differentiation of that cell type. They act as “co-incidence detectors” and make the binary decision of whether the gene should be expressed and by how much.

Question 9

What are the three broad classes of extracellular signals?

Answer 9

Ligand – receptor – intracellular cascade (exception is nuclear hormones). Paracrine, autocrine and endocrine.

Question 10

Paracrine extracellular signalling?

Answer 10

Produced locally to stimulate proliferation of a different cell type that has the appropriate cell surface receptor.

Question 11

Autocrine extracellular signalling?

Answer 11

Produced by a cell that also expresses the appropriate cell surface receptor.

Question 12

Endocrine extracellular signalling?

Answer 12

Like conventional hormones, released systemically for distant effects.

Question 13

What roles do extracellular signals play in cell growth and differentiation?

Answer 13

Proteins that: 1. stimulate proliferation and promote survival. Referred to as mitogens. E.g., growth factors and interleukins (EGF, FGF, NGF, PDGF, IGF1, IL2, IL4).

2. They can also induce differentiation and inhibit proliferation e.g., TGFbeta.
3. Or they can do either e.g., Wnt ligands – promote/inhibit cell growth. 4. Induce apoptosis e.g., TNFa and other members of the TNF family.

Question 14

How do extracellular signals induce gene expression?

Answer 14

Growth factor binds to its receptor on the outer surface of the cell. This activates a signal transduction pathway via a kinase cascade that activates transcription factors in the nucleus. These transcription factors will increase the expression of downstream genes, creating a mRNA which is exported back to the cytoplasm, where protein synthesis takes place. These proteins can remain in the cytoplasm where they exert their effect or return back to the nucleus to further increase expression of downstream genes.

Question 15

What is the M phase of the cell cycle?

Answer 15

Mitosis; cell divides into two daughter cells.

Question 16

What is interphase?

Answer 16

Having left mitosis, cell enters interphase which is the other three phases that are not mitosis. During this, cells grow in size as most macromolecules are synthesised continuously throughout interphase.

Question 17

What is the S phase of the cell cycle?

Answer 17

Opposite M phase; S = synthesis of DNA as DNA replication takes place here.

Question 18

What are quiescent cells?

Answer 18

Cell that has left cell cycle after mitosis. In stage G0. Can be in this stage for long periods of time. They can rejoint cell cycle to G1 to continue proliferating or they differentiate. Change shape to adopt a different function and ultimately, terminally differentiated into post-mitotic cells so cannot be more specialised. After some time, it will undergo apoptosis.

Question 19

How many chromosomes are there in a cell in G1 phase?

Answer 19

2N – diploid cell – 46 chromosomes

Question 20

How many chromosomes are there in a cell in G2 phase?

Answer 20

4N – tetraploid genome - 92 chromosomes

Question 21

How can we measure cell DNA content using FACS analysis?

Answer 21

Fluorescent flow cytometry. If a DNA stain is applied, FACs can measure the DNA content in every cell in a population. Date used to plot a graph – no. of cells vs amount of DNA.

Question 22

What is the difference between high and low rates of division when looking at FACS analysis graphs?

Answer 22

Typically get a graph where, as amount of DNA increases, you get a big peak of many cells which are in G1/G0 cells. The second peak is much smaller and are the tetraploid ones in G2/M. In between is S phase – have somewhere between diploid and tetraploid cells. This graph shows rate of division is low.

In a graph where rate of division is high, you can see cells in G1 phase has gone from 60% to 40%; cells in S phase have increased and G2/M % doesn’t increase much as it’s very time-limited part of cycle.

Question 23

What can we observe in fluorescence microscopy for each stage of the cell cycle?

Answer 23

Blue = DNA; red = y-tubulin; green = CHEK2; yellow = centrioles (both y-tubulin and CHEK2). Interphase = mainly blue and red. Prometaphase – cytokinesis = mainly blue and green.

Question 24

What are protein kinases?

Answer 24

Phosphorylate proteins and attach a phosphate molecule.

Question 25

What are phosphatases?

Answer 25

Remove phosphate or de-phosphorylate.

Question 26

Why are cell cycle checkpoints important?

Answer 26

They are controls to ensure the strict alternation of mitosis and DNA replication. Involve specific protein kinases and phosphatases.

Question 27

What are the requirements of a cell wanting to pass the restriction point (checkpoint)?

Answer 27

Key checkpoint since G0 will need to first pass through here. DNA not damaged, cell size, metabolite/nutrient stores.

Question 28

What are the requirements of a cell wanting to pass the G2-M phase checkpoint?

Answer 28

DNA completely replicated and DNA not damaged.

Question 29

What are the requirements of a cell wanting to pass the mitosis checkpoint?

Answer 29

Chromosomes physically aligned properly on spindle.

Question 30

Where do external signals (growth factors) act in the cell cycle?

Answer 30

They act in G1 phase, upstream of restriction point. Cells are responsive to growth factor and this is the main site of control for cell growth.

Question 31

What are CDKs and cyclins?

Answer 31

CDKs – catalytic subunit, 10 genes. Cyclin – regulatory subunit, more than 20 genes; expression induced by growth factors.

Question 32

How does the cyclin-CDK complex form?

Answer 32

When there are sufficient amounts of cyclin, it will form a complex with CDK. This active CDK-cyclin complex and it can bind and phosphorylate its specific substrate proteins.

Question 33

How is cyclin-CDK activity regulated?

Answer 33

1. Cycles of synthesis (gene expression) and destruction (by proteasome). 2. Regulation by post-translational modification by phosphorylation (of cyclins or CDKS) – may result in activation (increased kinase activity), inhibition or destruction (increased targeting to proteasome). 3. Regulation by dephosphorylation. 4. Binding of cyclin-dependent kinase inhibitors (CDKIs) (inhibits complexes).

Question 34

Explain the RB pathway.

Answer 34

Retinoblastoma protein (RB) is a key substrate of G1 and G1/S cyclin-dependent kinases. 1. Unphosphorylated RB binds E2F transcription factor preventing its stimulation of S-phase protein expression. 2. However, in the presence of cyclin D-CDK4 complex & cyclin E-CDK2 complex, RB becomes phosphorylated and dissociates from E2F. 3. E2F is no longer suppressed so it is able to bind to promoters of target genes. Released E2F stimulates expression of more cyclin E (creates a positive feedback loop) and S-phase proteins e.g., DNA polymerase, thymidine kinase, PCNA etc. DNA replication starts.

Question 35

Describe the full pathway of cell cycle regulation.

Answer 35

1. Triggered by mitogens acting in G1 phase that signal to the nucleus, via transcriptional activation, drive expression of early genes. 2. These early genes include transcription factors which drive expression of delayed genes. This includes cyclin D – which having increased expression forms active complexes with CDK4/6. 3. The activity of cyclin D – CDK4/6 active complexes are sufficient to hypo phosphorylate RB which allows some E2F activity such that there is some expression of cyclin E. 4. This gives rise to low levels of active cyclin E- CDK2 complex which causes hyper phosphorylation of RB. It has now lost all ability to repress E2F. 5. E2F activity remains high through next phases of cell cycle and therefore activation of E2F responsive genes. These are required for S phase. 6. Now, there is sequential activation of different cyclin-CDK complexes. So, cyclin E – CDK2 active complex is able to phosphorylate and activate cyclin A – CDK2, which does the same to cyclin A – CDK1 complex, which does the same for cyclin B – CDK1. 7. Final step of mitosis involves dephosphorylation of RB back to basal unphosphorylated state which is mediated by PP1.

Question 36

What happens if DNA is damaged when it’s checked at the checkpoint?

Answer 36

1. Stop of cell cycle – mainly driven by CDKI genes. 2. Attempt DNA repair – can re-enter cycle if repaired (nucleotide or base excision enzymes, mismatch repair etc.) 3. Apoptosis (BCL2 family, caspases).

Question 37

What does TP53/P53 stand for and what is its function?

Answer 37

Tumour protein 53. It is a tumour suppressor gene.

Question 38

What is TP53 role in DNA damage?

Answer 38

If normal DNA, TP53 is continuously destroyed by proteasome and hence, there are low levels. If DNA becomes damaged, (e.g., mutagen binds to it), it’s identified and this leads to kinase activation including DNA-dependent kinases. These phosphorylate TP53 which means it can no longer be destroyed by proteasome. TP53 levels increase and exerts its effects on cell.

Question 39

What are the biological effects of phosphorylated TP53 on the cell?

Answer 39

1. Expression of CDKI: cell cycle arrest. 2. DNA repair e.g., excision repair. 3. Repair not possible: apoptosis.

Question 40

How is TP53 involved in cancer?

Answer 40

TP53 loss-of-function mutations are amongst the most frequent in cancer. They prevent cell cycle arrest – hence faster growth. They prevent apoptosis – hence they do not die. They prevent DNA repair – hence more mutations which causes more heterogeneity, more chance of adapting, increased cancer progression.

Question 41

How do traditional chemotherapeutic drugs affect the cell cycle?

Answer 41

Stop proliferation and induce apoptosis.

Question 42

How do S-phase drugs help treat cancer?

Answer 42

They cause DNA damage e.g., 5-fluorouracil (prevents synthesis of thymidine) and cisplatin (binds to DNA causing damage and blocking repair).

Question 43

How do M-phase drugs help treat cancer?

Answer 43

Target the mitotic spindle. E.g., vinca alkaloids – stabilises free monomeric tubulin, prevents microtubule polymerisation and arrest cells in mitosis. Paclitaxel (Taxol) – stabilise multimeric complexes of microtubules and prevent de-polymerisation. This is opposite to alkaloids but still arrests cell in mitosis.