Unit 4- Control of Cell Cycle & Cell Death Flashcards
How is the cell cycle regulated?
- external signals influence the cell cycle clock
Anti-Mitogenic signal > enter G0/ quiescent phase
Pro-Mitogenic signal > enter active cell cycle (G1>S>G2>M)
What are the phases of the mammalian cell cycle?
G0- Non-proliferative/ Quiescent phase
G1- Proliferation decision
S- DNA replication
G2- Preparation for mitosis
M- Mitosis
What are the cell cycle checkpoints?
Start Checkpoint- is environment favourable > proceed to S phase
G2/M Checkpoint- is all DNA replicated > enter mitosis
What does cell cycle progression rely on?
- protein presence
- protein localization (cytoplasm vs nucleus)
- protein activation status (phosphorylated ext)
What are cyclins?
- cell cycle proteins that bind to CDKs to activate them/ modulate phosphorylating ability
- undergo cyclic destruction/ synthesis
What are the types of cyclins?
G1 Cyclins- bind CDKs at G1/ promote entry into S phase
Mitotic Cyclins- bind CDKs at G2/ promote entry into M
What are CDKS?
CDK= Cyclin Dependent Kinase
- phosphorylate substrates on serine/ threonine residues
What are CKIs?
CKI = Cyclin-Dependent Kinase Inhibitor
- interfere with Cyclin/CDK complexes (binding/ phosphorylation)
What are the 2 families of CKIs?
Cip/Kip- involved in G1 and G1/S control
Ink4- involved in G1/S control
How do tumor suppressor/ oncogene mutations cause cancer?
Tumor Suppressor mutation > loss of function (brakes)
Oncogene mutation > gain of function (growth)
How is progression through cell cycle checkpoints regulated?
- regulated by CDKs
- depend on regulatory subunit cyclin for activity
- different CDK/cyclin pairs throughout cell cycle > different substrates
> cyclin levels change rapidly throughout cell cycle (1 direction cycle)
What ensures the cell cycle occurs in only 1 direction?
- rapid changes in cyclin levels throughout the cell cycle
How are cyclin LEVELS regulated?
- Ubiquitin ligase enzyme promotes ubiquitylation
- adds ubiquitin to phosphorylated cyclins
> targets cyclin protein for degradation by proteasome - ensures rapid/ regulated reduction in CDK activity
How is cyclin ACTIVITY regulated?
- CDK/cyclin complexes are functionally regulated by CKIs
INK4- regulate cyclin D complex (G1)
Cip/Kip- regulate other cyclin complexes
How can CKIs regulate growth inhibition/ growth promotion?
- inhibitory factors like TGFβ trigger ↑ expression of CKIs
> growth inhibition - mitogens phosphorylate CKIs > phosphorylated CKIs can not translocate to nucleus/ inhibit CDK-cyclin complexes
> growth promotion
What is the period where cells are responsive to mitogenic GFs/ inhibitory TGFβ?
G1 (up until R point)
What is the R-point?
Restriction Point- once cell passes, fully commits to cell cycle
- late in G1
- cell commits to a) advance through cell cycle/ b) remain in G1/ c) retreat from active cell cycle into G0
- deregulation of R-point in many cancers
What drives cells through the restriction point?
Pocket Proteins = RB Retinoblastoma proteins
- tumor suppressors (lack of functional gene > eye tumors)
- phosphorylation state of pRb influences activity
- hyperphosphorylation of pRB required for transition through R point
- pRb binds to E2F family transcription factors/ inhibit activity
How does the phosphorylation status of pRb (pocket protein) influence the cell cycle?
Hyperphosphorylated pRb = inactive/ can not bind and inhibit E2F transcription factors
- cyclin activity leads to ↑ pRB phosphorylation > bypass G1 R-point
- pRb becomes more phosphorylated as cell cycle progresses
Early-mid G1- unphosphorylated > hypophosphorylated
Late GI- hypophosphorylated > hyperphosphorylated
How is the R point perturbated in cancer?
- oncogenes promote advancement through restriction point ex) Myc
- tumor suppressors block advancement through restriction point
What are the steps in DNA damage repair?
- DNA lesion detection
- Recruitment of DNA-repair factors
- Activation of DNA-repair enzymes
- Repair
- Degradation/ inactivation of DNA repair factors
What are the potential consequences of DNA damage in cell cycle?
Repair/ Apoptosis/ Senescence/ Mutation
What is a drug developed to target the cell cycle?
Palbociclib = selective CD4/6 small molecule inhibitor
- initial attempts to target CDKs failed due to broad actions against all CDKS (poor specificity > high toxicity)
- RB deficient tumors do not respond/ tumors can become resistant
What is the difference between Necrosis/ Apoptosis?
Necrosis- pathological/ uncontrolled/ induced by ROS, trauma/ tissue damage/ neighbouring cells affected/ membrane disruption/ cell swells
Apoptosis- controlled cell death/ no inflammation/ no tissue damage/ surrounding cells normal/ induced by specific signaling or cell stress
What are 3 apoptosis assays?
- caspases are key molecules in apoptosis
Phosphatidylserine/ Annexin V staining
- recognition signal for macrophage engulfment
TUNEL Assay
- addition of labeled nucleotides > nicked DNA
- not specific to apoptosis (labels any fragmented DNA)
Immunostaining for activated Caspase 3
- requires antibodies that only recognize caspase 3
What are the 2 major apoptosis pathways?
Intrinsic (mitochondrial) / Extrinsic (death receptor)
- converge at execution phase of apoptosis
What are the 2 types of caspases?
Initiator- detect/ transduce signals/ activate effector caspases/ protein-protein interactions
Effector = Executioners- cleave downstream targets > cell death
What is the caspase paradox?
- caspases cleave substrates at Asp-X-X-Asp
- caspase activation requires cleavage at Asp-X-X-Asp
What is APAF-1?
- assembles into apoptosome (wheel of death) to activate caspases
- several domains including CARD for protein interactions
How are apoptosomes formed?
- Cytochrome C interacts with bound ATP, inducing hydrolysis to ADP
- results in conformational change in APAF-1
> exposure of CARD domain > interact with each other > complex forms
(many APAF-1s come together to form an apoptosome) - ADP/ATP swap allows APAF-1 molecules to assemble > apoptosome by binding ar CARD domains
How does caspase activation work?
- procaspase 9 binds via CARD to apoptosome
- more CARD bound procaspase 9’s recruited > antiparallel orientation
- leads to autoactivation of apoptosome-bound caspase 9
> caspase cascade leading to apoptosis
How does Cytochrome C induce apoptosis?
- cytochrome c sequestered in intermembrane space of mitochondria
- release triggered by pro-apoptotic events > leads to activation of procaspase 9 ext
- cytochrome c release also occurs with mitochondrial damage
What is the Fas/FasL pathway?
- Fas ligand binds to receptor > assembly of DISC
> activation/cleavage of procaspase 8/10 > activation of executioners
How is procaspase 8 activated?
- initial ligand-receptor interactions > clustering
- recruitment of FADD proteins > large clusters of FAS/FASL
> DISC assembly (DISC is like apoptosome) > activate procaspases
How is the mitochondrial pathway activated?
- active caspase 8 > cleavage of Bid
- truncated Bid > induces cytochrome c release from mitochondria
- cytochrome c enhances activation of effector caspases
What are the 2 main protein groups of Bcl-2 family?
Pro-Survival > Bcl-2/ Bcl-x
- maintain integrity of mitochondrial membrane/ prevent cytochrome c release (protect cells from apoptosis)
Pro-Apoptotic> Bax/ Bak/ Bad/ Bim/ Noxa/ Puma
- promote cytochrome c release from mitochondria (tumor suppressors)
What is the only cytotoxic/pore-forming Bcl-2 protein?
Bax members (Bax/Bak)
How do Bcl-2 proteins work/ how do they promote apoptosis?
Bax/Bak = pore-forming (pro-apoptotic)
- actively inhibited by Bcl-2/Bcl-x (pro-survival)
BH3 Only family (pro-apoptotic Bcl-2’s not including Bax/Bak inhibit prosurvival members (Bcl-2/Bcl-x)
- inhibition of inhibitors > pro-apoptotic events
How are BH3 only proteins regulated?
Bim/ Bid/ Bad > Post-translational mechanisms
Puma/ Noxa > De novo expression
- gene expression controlled by p53
How does p53 contribute to cancer?
- master transcription factor, gene promoters contain p53 binding sites
- loss of function of p53 rather than loss of protein (mutations)
- pro-apoptotic protein prone to inactivation
How does p53 induce apoptosis?
- Fas production > activation of procaspase 8
- Bax production > ↑ release of cytochrome c from mitochondria
What is a question about p53?
- is main oncogenic action via interference with p63/p73?
(mutant p53 interferes with p63/p73 binding to gene promoters)
What is anoikis?
- loss of ECM (cell: matrix) / cell: cell adhesion > metastasis
- PKB-AKT signaling has key role in cell death
What is the UPR?
- unfolded protein response > to reduce ER stress
- if persists can trigger apoptosis/ mutation
