Blair Cell Cycle Flashcards
Where do quiescent cells reside?
G0/G1 phase
How long is DNA synthesis phase?
S phase, 10 hours
Which cell phase is most manipulative?
G1 phase ~9 hours
Which cells are slower through G1 phase: Stem cells or differentiated cells?
Stem (primary) cells
What does colemid do to allow study?
binds mictrobules to inhibit mitosis and enrich M phase
Which cell phase is visible under a light microscope?
M phase
What do hydroxyurea and thymidine inhibit?
Polymerase in S phase
Which base analogues can S phase be detected with?
bromodeoxyuridine replaces thymidine detected with anti-BUdR antibodies
Which phases cannot be separated by FACS?
G2 and M (4n diploid genome)
Where were cdc genes discovered?
yeast cells
Which transition does MPF control?
G2/M
Which MPF component is constitutively expressed?
cdk1
What is the cdc2 analogue in mammals?
cdk1
What is MPF made up from?
Cyclin B regultory subunit and cdk1
What does wee1 do to cdk1?
phosphorylate Y15 to block ATP binding
Which proteins have antagonistic action on Y15?
wee1 and cdc25
What does cak do to cdk1?
primes in G2 by phosphorylation of T161 for substrate binding.
What does active cdk1 do?
phosphorylates condensin for chromatin condensation, spindle formation, ER and golgi fragmentation, lamina breakdown
What does anaphase promoting factor do?
E3 ubiquitin ligase for degradation of cyclin B after mitosis has started, degrades securin to separate sister chromatids
Which cdk/cyclin complex controls G1?
cdk4/cyclin D
Which checkpoint is for integrity of DNA?
G1/S by cdk2/cyclin E. E2F tf bound by Rb and HDACs for repression of transcription, released by Rb hyperphosphorylation in late G1. Transcription of S phase complex genes cdk2/cyclin A
Which genes control G1/S transition?
cdk2/cyclin E
Which genes control S phase checkpoint?
cyclin A/ cdk2 transcribed by E2F
Where was MPF discovered?
sea urchins
Where does positive feedback of MFP originate?
cdc25
What feedback does wee1 produce?
negative feedback to MPF
What are the CDK inhibitor families?
Cdk Inhibitory Proteins (CIP), Inhibitor of cdk4 (INK)
Which proteins belong to CIP?
p21, p27, p57
Which proteins belong to INK?
p16
How do CIPs initiate DNA damage responses?
DNA damage caused ATM kinase to phosphorylate p53. MDM2 ubiquintatin ligase targetting inhibited so p21 transcription activated. Cells arrest in G1.
What does p53 control?
apoptosis. mutation results in cancer
Which stimuli act on G0 cells?
EGF, PDGF for RTKs; GPCRs for cAMP/PKA for nuclear transcription factors c-Myc, c-Jun, c-Fos
What is apoptosis?
Programmed cell death in an orderly and controlled manner with a defined programme
What is TUNEL assay?
Fluorescent labelling of DNA fragment termini
What does Anexin V label?
Apoptotic phospholipid membrane receptors for FITC quantification
How can apoptosis be observed?
Anexin V FITC, TUNEL, DNA ladders, cell morphology, western blotting of caspases
When are early response genes overactivated?
cancer
Are early response genes maintained?
No, they decline after activating expression of delayed response genes
Does synthesis of early response genes require proteins?
No
What are caspases?
Cys-catalysed ASP-targetting proteASES
How are caspases activated?
Cleavage of N terminal regulatory domain, heterodimerisation
What are the 2 classes of caspases?
Initiator (8&10, 9), executioner (7&3)
Which initiator caspases are activated by the intrinsic pathway?
9
Which initiator caspases are activated by the extrinsic pathway?
8 & 10
What does c-FLIP do?
Inhibit caspase 8 activating executioners
When is c-FLIP overexpressed?
In death-ligand resistant cancers
How are caspases activated?
Cleavage of N terminal regulatory domain, small and large domains; heterodimerisation
Examples of intracellular/intrinsic decision pathway
p53 upregulation, DNA damage, hypoxia, withdrawal of survival factors, UV radiation
Which death receptors are involved in extrinsic exectution phase?
TRAIL acts on death receptors 4/5
FasL on Fas
TNFalpha on TNFR-1
What are the 2 phases of apoptosis?
decision and exectution
What is the extrinsic exectution pathway?
Ligand binds, homotrimerisation, recruitment of FADD through death domain, acts as an adapter to recruit caspases 8 & 10 through Death Effector Domain to assemble DISC and cleave/ activate caspases
How do cells become resistant to extrinsic death ligands?
soluble or membrane bound decoy receptors, intracellular signalling inhibitors
What is intrinsic execution pathway?
Stimulus releases cytochrome C from mitochondria. Binds Apaf1: hydrolysis of dATP and activation of CARD domain. dADP-dATP exchange triggers assembly of apoptosome. CARD domain recruits propase 9 inititator.
Which pathway does BCL-2 family operate in?
Instrinsic
What does BCL BH3 do?
Block BH1234
What does BCL family BH1234 do?
Block aggregation and pore formation of BH123
Which molecules are BCL BH123?
pro-apoptotic
What action are BCL BH1234?
antiapoptotic
Which action is BCL BH3?
proapoptotic
Which BCL proteins does p53 upregulate?
Bax (B) and Puma (C) to promote apoptosis
Which pathway doe IAPs block?
Apoptotic intrinsic pathway by binding caspases
How are IAPs counteracted?
anti-IAPS released through BH123 pores
How long are telomeres?
12kb/ 2 thousand repeats
What is the sequence of telomeres?
TTAGGG
How long is the telomere 3’ overhang?
100-200nt
What is the purpose of the telomere 3’ overhang?
forms T loop to bind proteins to pair sister chromatids and project against shortening and entanglement
How many bases are lost from a telomere in mitosis?
100bp
Where does telomerase act?
germline and stem cells, cancer and unicellular eukaryotes
How does telomerase act?
Removal of primer, TERC template binds for TERT synthesis. Ratchet mechanism
What does TERC consist of?
451nt AAUCCC
What does shortening of telomeres cause?
Senescence-aging and death after ~ 50 divisions
