Mechanisms of disease

Question 1

How can the cell cycle be visualised/quantified?

Answer 1

visualised via fluorescence microscopy which generates stains of proteins.

quantified via fluorescent flow cytometry which measures the DNA content of every cell population

Question 2

How is the cell cycle regulated?

Answer 2

Regulated via cyclin-dependent kinases (CDK) which form a complex with cyclin, causing its activation. It then phosphorylates a specific substrate protein.

This is regulated by:

1. continuous cycles of synthesis/apoptosis
2. post-translational modification via phosph.
3. dephosphorylation
4. binding of CDKI

Question 3

What are cyclins?

Answer 3

encoded by 20 genes, cyclins are regulatory subunits controlled by GF

Question 4

What are CDKs?

Answer 4

encoded by 10 genes, CDKs are catalytic subunit

Question 5

Explain the mechanism of retinoblastoma protein .

Answer 5

1/ unphosphorylated Rb binds to E2F transcription factors, preventing expression of S-phase proteins
2/ in presence of cyclin D-CDK4 or cyclin E-CKD2, Rb becomes phosphorylated and drives replication. It also stimulates expression of more cyclin E, creating a positive feedback.

Question 6

Explain the regulation of cell cycle and apoptosis.

Answer 6

1. GF drive early gene expression - TF FOS, JUN, MYC
2. This stimulates delayed gene expression
3. E2F sequestered by binding to unphosphorylated RB
4. G1 cyclin-CDK complexes hypophosphorylate Rb allowing more E2F activity + more cyclin E, S-phase proteins
5. G1/S phase cyclin-CDK complexes hyperphosphorylate Rb - stimulating release of E2F

Switches activated by post-translational modification or removal of inhibitors

Question 7

Explain mechanisms by which growth control can be disrupted in neoplasia

Answer 7

TP53 is continually destroyed by proteasome.

Mutagen binding induce activation of protein kinases which phosphorylate TP53 preventing its destruction to:

1. drive expression of CDK (malfx: cells proliferate faster)
2. activates DNA repair mechanism (malfx: more mutations)
3. induce apoptosis (malfx: become resistant to dying)

Question 8

Define drugs acting on the cell cycle: S-phase drugs

Answer 8

they cause DNA damage

1. 5-fluorouracil - prevent thymidine synthesis
2. cisplatin - binds to DNA to cause damage and block repair

Question 9

Define drugs acting on the cell cycle: M-phase drugs

Answer 9

they target the mitotic spindle:
vinca alkaloids, paclitaxel :
- stabilises microtubules
- prevent polymerisation
- arrest cells in mitosis

Question 10

Define the response of the cell in response to DNA damage

Answer 10

may trigger cell cycle arrest or apoptosis:

1. stop the cell cycle - CDKI genes, CHEK2, etc.
2. DNA repair - nucleotide/base excision enzymes, mismatch repair, etc.
3. programmed cell death - BLC family, caspases