What does propidium iodide do?
Interchelates in DNA to allow you to see amount of DNA in flow cytometry
Major cyclins and their formation of cyclin-cdk complex during which cell cycle?
- G1 (cyclinD-cdk4,6)
- G1/S (cyclinE-cdk2)
- S (cyclinA-cdk2)
- M (cyclinB-cdk1)
** Cyclin D is major cyclin controlling G1 progression to S
Two classes of CDK inhibitors?
INK4: binary, bind to CDK and prevent binding to cyclin to inhibit it
CIP/KIP: ternary, bind to Cylcin-CDK complex
Cell cycle checkpoints?
End of G1: is DNA damaged? (p53 checks this)
End of S: Is all DNA replicated
End of G2: Is cell big enough? Prevents initiation of mitosis until DNA replication is complete
In M: Are all chromosomes appropraitely aligned on spindle?
With Rb protein how many alleles must be lost? What restriction point does it work in?
BOTH alleles must be lost; End of G1 restriction point
p53 pathway of cytostatic and cytotoxic mechanism?
: low level DNA damage induces low level of p53 expression induces p21 (CIP/KIP ternary CDK-inhibitor) which blocks G1/S progression
- Cytotoxic pathway: unrepairable DNA damage induces high expression of p53 which transcribes Bax who blocks Bcl2, allowing the mitochondria to release cytochrome c. Cytochrome c binds to APAF (apoptosis protease activating factor) and pro-caspase 9 to form apoptosome. Apoptosome activates procaspase 9 to caspase 9.
Name the initiator caspases and executioner caspases?
Initiator caspases: 8,9,10
**caspases (cystein-aspartate protease) are pro-enzymes that need to be cleaved to become activated, can activate endonucleases that cleave DNA, can cleave lamin
Pro- or anti- apoptotic Bax, Bid, Bcl2?
Bax, Bid: Pro-apoptotic
Apoptotic vs. Necrotic cell?
Apoptotic: membranes remain intact until the very end (Trypan blue can show that the cells dying by apoptosis are at end-stage). Cells shrink, membrane intact until the end, dna degrade and stick to walls. Phosphotidylserine is flipped to outer leaflet during apoptosis and Annexin V can be used to check for apoptosis. Does not set-up inflammatory response in body. Caspases degrade DNA in apoptotic cells in the linker region (around H1).
Necrosis: cell-membrane compromised, cell contents spilled outside, causes inflammatory response
Goes through cell membrane of dead cells (i.e. necrotic cells or late-phase apoptotic cells) to ID them as dead.
Cancer drug used to inhibit Topoisomerase II
Cancer drug used to inhibit Topoisomerase I
How to tell if cells are undergoing apoptosis?
- Flow cytometry (if you see a region in the sub-G1 section)
Extrinsic pathway of apoptosis?
- Trail binds DR4/DR5 (which trimerize) --> form DISC --> induces FADD --> Activates Caspase 8,10 --> activates caspases 3, 6, and/or 7. FLIP blocks caspase 8,10 activation (i.e. doesn't induce apoptosis). FLIP is upregulated in cancer cells.
There is crosstalk between intrinsic (i.e. cytochrome c) and extrinsic (DISC, FADD) pathways.
Cross talk between restriction point and check point (i.e. p53 and Rb).
if Rb is lost, E2F1 is free, it will drive the transcription of ARF gene which blocks MDM2 (a ligase that puts the ubiquitan on p53)... and an increase in p53 kills cells