cell cycle, cell death and cancer Flashcards
what are positive vs negative regulators of the cell cycle
Positive: each cyclin acts at a specific stage of the cell cycle and sets up the transition to the next stage, then gets degraded
Negative: surveillance mechanisms check for the proper progression through the cell cycle, if anything goes wrong checkpoints hold the cell cycle until repair is done
what are the cell cycle checkpoints in mitosis
spindle assembly checkpoint: are all the chromosomes attached to mitotic spindles? (prophase)
chromosome segregation checkpoint: have all chromosomes reached opposite poles (after anaphase)
what is cell synchrony
synchronizing cells to study the cell cycle
- hydroxyurea of thymidine inhibit the synthesis of dNTPs and cells arrest in early S phase
- Nocodazole disrupts the mitotic spindle and cells arrest in pro-mretaphase
- washing away the agent allows for simultaneous progression of all cells through the cell cycle
What does flow cytometry measure and how does it do it?
measures the amount of cellular DNA - doesn’t require a synchronized cell culture
- 1C DNA content = unreplicated, most cells found here
- 2C DNA content = replicated, less than half the cells
What did the discovery of cyclins in dividing eggs conclude
- for proteins that are continuously synthesized and periodically destroyed, cyclin levels rise during interphase, peak at mitosis and drop before anaphase
What to G1 cyclins (CycD / CDK4/6) regulate
- coordinates entry into a new cell cycle in response to growth factors
- inactivate CDK inhibitors allowing the activation of C1/S cyclins
what do G1/S cyclins (CycE / CDK2) regulate
- inactivate inhibitors (Rb) of entry into S-phase
- stimulate synthesis of genes controlling S phase progression including cyclin A
what do S phase (CycA / CDK2) regulate
- irreversibly initiate DNA replication
what do M cyclins (CycB / CDK1) regulate
- stimulate assembly of the mitotic spindle, chromatin condensation, nuclear envelope breakdown
- destruction in anaphase by the APC leads to mitotic exit
what are the regulators of cyclin-CDK activity
- cyclins
- kinases and phosphotases
- inhibitory proteins
- ubiquitin-protein ligases
how are CDKs regulated
regulated by activation and inhibiting their phosphorylation
- phosphorylation of a threonine residue near the active site of the CDK is mediated by CAK, happens as soon as the cyclin-CDK complex is formed
- Cdc25 is a phosphatase, dephosphorylation of CDKs regulates G1->S and G2->M transitions
Cyclin and CDK inhibitors: G1/S transition
- G1/S CDKs activate the expression of S phase cyclin CDK components
- G1/S CDKs phosphorylate S phase inhibitors so it can be activated
- SCF proteasome degrades the phosphorylated S phase CDK inhibitor to allow the cyclin to to bind to S phase CDKs
Cyclin degradation: mitosis
- APC/C-Cdc20 proteasome degrades securin in the metaphase->anaphase transition
- in mitotic exit, phosphatase activates Cdh1 and APC/C -Cdh1 proteasome degrades mitotic cyclins
how is the G1/S phase transition controlled in mammals
- In early G1 Rb is bound to the transcription factor E2F suppressing genes required for S-phase
- Growth factors and signal transduction stimulates CyclinD-CDK4/6 expression
- E2F stimulates the transcription of cyclin E/A-CDK2
- Cyclin E-CDK2 further phosphorylates Rb resulting in commitment to pass the restriction point: cell enters S phase and centrosome duplication happens
How is DNA replication initiated in G1
through loading of ORC and MCM-helicase
- all mitotic cyclins are degraded (from M phase)
- ORC binds to DNA origin, Cdc6 and Cdt1 load inactive MCM-helicase to ORC to form the pre-RC
- now in a low CDK state
