2. MECHANISMS OF DISEASE I - CELL GROWTH & DIFFERENTIATION

Question 1

What’s the difference between cell growth & cell differentiation?

Answer 1

• Both cell differentiation & cell growth are processes that are involved in turning a zygote into a mature organism

Question 2

What are the three main groups of disease related to cell growth & differentiation?

Answer 2

1. DEVELOPMENTAL CONDITIONS
   - defects related to cell growth and/or differentiation
   - e.g neural tube defects
2. NEOPLASIA & METAPLASIA
   - Neoplasia = uncontrolled cell growth
   - Metaplasia = transformation of a cell from one type into another
   - e.g tumours & cancers
3. OTHER
   - e.g cardiac hypertrophy

Question 3

What are the two main forms of cell growth?

Answer 3

1. Hypertrophy

2. Hyperplasia

Question 4

What is hypertrophy?

Answer 4

• An increasein size, cells become bigger
• More proteins, lipids, nutrients etc.
• Increased protein synthesis is sufficient to cause increase in cell size

Question 5

What is hyperplasia?

Answer 5

• An increase in celll number due to more cells
• Hyperplasia mainly caused by cell proliferation & cell division
• Hyperplasia is more common than hypertrophy

Question 6

How do extracellular signals interactwith the promoter?

Answer 6

• Cell differentiation & cell growth are controlled by the integartion/combination of intracellular & extracellular signals
• These signals converge onto the rpomter of a key gene involved in cell proliferation & differentiation
• Promoters of these genes act as co-incidence detectors, decision can be made about whetehr or not to express gene & how much

Question 7

What are the three classes of extracellular signals?

Answer 7

1. PARACRINE - secreted locally (e.g neighbouring cell) which causes proliferation of different cell type
2. AUTOCRINE - cell secretes it’s pwn ligand which it can then bind to
3. ENDOCRINE - released systemically/ into the circulation for distant effects

Question 8

What are mitogens & give examples?

Answer 8

• Mitogens are extracellular signals that stimulate proliferation & promte cell survival
• E.g growth factors (EGF, FGF), interleukins

Question 9

Give an example of a signal which can inhibit & stimulate proliferation?

Answer 9

• TNF Beta

Question 10

Give an example of a signal which can inhibit/stimulate proliferation & promote differentiation?

Answer 10

• Wnt ligands

Question 11

What can TNF alpha induce?

Answer 11

• TNF alpha can induce apoptosis

Question 12

Describe the process by which extracellular signals induce gene expression

Answer 12

1. Extracellular signal (GF) binds to receptor
2. Activates signal trasnduction pathway
3. Transcripton factors are activated via kinase cascade
4. mRNA transcribed & proteins produced
5. Proteins can remain in the cytoplasm or membrane, or return to nucleus to act as TF or nuclear protein

Question 13

What are the 4 phases of the cell cycle?

Answer 13

1. G1 PHASE - Growth phase, preparation for S phase. Responsive to Growth factors
2. S PHASE - DNA synthesis occurs & chromosome replication
3. G2 PHASE - growth & preparation for M phase, organelles & cytoplasm doubled
4. M PHASE - Mitosis & cytokinesis
5. G0 PHASE - rets phase where no cell division oocurs, state of quiescence
   - G0 & G1 phase = didploid
   - S phase = in between diploid & tetrapolid as cells are still replicating
   - G2 & M phase = after replication - tetraploid

Question 14

What are the 5 phases of mitosis?

Answer 14

1. PROPHASE - chromosmes condense, nuclues becomes less visible. Centrioles migrate to opposite poles & mitotic spindles form
2. PROMETAPHASE - nuclear envelope breaks down & centromere attaches to mitotic spindle via kinetochore
3. METAPHASE - chromosomes are at their most condensed, chromosomes align on equator known as metaphase plate, centrioles at opposite ends
4. ANAPHASE - ssister chromatids sepaarte synchronously, daughter chromsomes migrate to oppsite poles
5. TELOPHASE - chromsomes arrive at centrioles & decidense, nuclear envelope reforms
   - Cytokinesis - cytoplasmic division of daughter cells

Question 15

How do cells become terminally differentiated?

Answer 15

• Cells leave the cell cycle and are termed post-mitotic cells
• Cell type specific gene expression results in changes in cell morphology & function

Question 16

What are the two outcomes of quiesecent cells?

Answer 16

• Quiescent cells have entered G0 phase meaning they no longer undergo cell division, so can have two outcomes:
  1. Terminally differentiate
  2. Re-enter cell cycle
• Ultimate fate of cells is apoptosis

Question 17

What can flow cytometry show about DNA content?

Answer 17

• Rate of division determined by number of cells in G1 relative to G2/M phase
• G1 = diploid, G2/M phase = tetraploid
• High n in G1 relative to G2/M = low rate of division
• Lower/ equal n of cells in G1 relative to G2/M = high rate of division

Question 18

What are cell cycle checkpoints?

Answer 18

• Cell cycle checkpoints are a strict set of controls which controls the alternation between DNA replication & mitosis

Question 19

What are the three cell cycle checkpoints?

Answer 19

1. G1 CHECKPOINT (end of G1 before S phase)
   - Known as the restriction point, considered to be teh msot important.
   - Checks for DNA damage, metabollites & nutrients stores to ensure everything correct before replication
2. G2 PHASE (end of G2 before M phase)
   - Checks DNA is correctly replicated to ensure enough chromsomal DNA for two daughter cells. Checks DNA damage before mitosis
3. M PHASE CHECKPOINT
   - Checks chromosomes are correctly alligned on the spindle before metapahse to ensure correct number of chromsomes in daughter cells

Question 20

What are cyclin dependent kinases?

Answer 20

• Cyclin dependent kinases (CDKs) are catalytic sub-units encoded by 10 gens.
  CDK must be bound to cyclins to be active & phosphorylate substrates of target proteins

Question 21

What are cyclins?

Answer 21

• Cyclins are reguatory sub-units made up of more than 20 gens
• Cyclins control the activation of CDKS
• Different cyclins are produced durig different phases of the cell cycle & they bind to their corresponding CDKs
• When there’s sufficient cyclin expression, it will form a complex with CDKS

Question 22

How is cyclin-CDK activity regulated?

Answer 22

• Cyclins have a high rate of turnover, being degraded by the proteasome
• Cyclins & CDKS can be phsophorylated or dephosphorylated, eitehr activating or inhibiting them
• Binding of cyclin dependent kinase inhibitirs (CDI) to CDKs to cause cell cycle arrest

Question 23

What is retinoblastoma?

Answer 23

• Retinoblastoma is a tumour suppressor gene
• It’s a key substrate of G1/S phase CDKs
• When unphosphorylated, retinoblastoma supresses E2F

Question 24

What is E2F?

Answer 24

• E2F is a transcription factor involved in the trasncription of S phase related proteins such as DNA polymerase, thymidine kinase etc.
• When retinoblastoma binds E2F, it prevents E2F from binding to the promoter of S phase related proteins meaning there’s no progression into the S phase

Question 25

Describe how retinoblastoma is phosphorylated

Answer 25

• When retinoblastoma is phosphorylated, it cannot bind E2F & therefore no longer acts as a TF
• Cyclin D-CDK 4/6 hypophosphorylates retionblastoma, allowing small E2F activity
• E2F results in Cyclin E production, which then forms a complex with CDK 2
• Cyclin E-CDK 2 hyperphsophorylates retionoblastoma, releasing E2F
• There is positive feedback for Cyclin E & E2F, as E2F activity increases, more cyclin E is produced which in turn results in more E2F activity

Question 26

Describe the sequence of events in the cell cycle involving retinoblastoma

Answer 26

1. Mitogens/growth factors act on G1 phase to cause EARLY GENE EXPRESSION
2. EARLY GENE EXPRESSION causes expression of DELAYED GENES such as Cyclin D, CDK 4/6 & E2F
3. Cyclin D levels increase and forms a complex with CDK 4/6.
4. Cyclin D-CDK 4/6 HYPOPHOSPHORYLATES retinoblastoma, allowing some E2F activity
5. Small amounts of E2F activity result in Cyclin E expression
6. Cyclin E forms a complex with CDK 2.
7. Cyclin E-CDK 2 HYPERPHOSPHORYLATES retinoblastoma, releasing E2F
8. E2F activity remains high resulting in expression of S phase proteins, allowing progession into the S phase
9. Sequential activation of cyclin-CDK complexes
   Cyclin E-CDK2 -> Cyclin A-CDK2 -> Cyclin A-CDK1 -> Cyclin B-CDK1
10. Dephosphorylation of retinoblastoma by PP1

Question 27

What are the three responses to DNA damage?

Answer 27

1. Cell cycle arrest - principally driven by CDK1 but also CHEK2
2. DNA Repair - base excision, mismatch repair
3. Apoptosis - driven by BCL2 & caspases

Question 28

What are the different cyclin-CDK complexes of the cell cycle?

Answer 28

• G1 PHASE = Cyclin D-CDK 4/6
• G1/S PHASE = Cyclin E-CDK2
• S PHASE = Cyclin A-CDK2
• G2 PHASE = Cyclin A-CDK1
• M PHASE = Cyclin B-CDK1

Question 29

What is TP53?

Answer 29

• TP53 is the tumour suppressor gene which is considered to be the ‘guardian of the genome’
• Normally, TP53 levels are low or virtually zero due to degradation by the proteasome but it can be activated in response to DNA damage

Question 30

How does DNA damage result in TP53 activation?

Answer 30

1. Mutagens/DNA damage cause kinase activation
2. Kinases phosphorylate P53
3. Phosphorylated P53 cannot be degraded/broken down by proteasomes

Question 31

What are the three effects of TP53 in response to DNA damage?

Answer 31

1. Cell cycle arrest - through CDKI expression
2. DNA repair - excision repair
3. Apoptosis (if repair is not possible)

Question 32

What are the consequences of P53 mutations?

Answer 32

• P53 is a tumour suppressor gene, so mutations can lead to a loss of function. P53 is no longer able to control cell proliferation & cannot suppress tumour debvelopment resulting in cancer
• Loss of P53 mutation+
  1. NO CELL CYCLE ARREST = Faster growth/proliferation
  2. NO DNA REPAIR = Cancer cells accumulate mutaions and adapt to environment allowing cancer to progress
  3. NO APOPTOSIS = cells don’t die

Question 33

What are the two classes of chemotherapeutic drugs that act on the cell cycle used to treat cancer?

Answer 33

1. S phase drugs

2. M phase drugs

Question 34

What are two examples of S phase drugs?

Answer 34

• S phase drugs cause DNA damage so that the cells cannot progress into the S phase because of the G1/S phase checkpoint
  1. 5-FLUOROURACIL - prevents synthesis of thymidine
  2. CISPLATIN - binds to DNA causing DNA damage & blocks DNA repair

Question 35

What are two examples of M phase drugs?

Answer 35

• M phase drugs target the mitotic spindle, so that the centromere cannot attach properly to mitotic spindle
  1. VINCA ALKALOIDS
  2. PACLITAXEL

Question 36

What is Vinca Alkaloid?

Answer 36

• Vinca Alkaloids stabilise & binds to free/monomeric tubulin
• PREVENT POLYMERISATION so that microtubules cannot form = no mitotic spindle formation
• Cells are arrested in mitosis

Question 37

What is Paclitaxel?

Answer 37

• Paclitaxel stabilises microtubules/polymerised tubulin
• PREVENTS DEPOLYMERISATION of microtubules
• Cells arrested in mitosis