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42 Cards in this Set
- Front
- Back
What is the purpose of flow cytometry?
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To determine the number of cells in G1, S, G2-M.
Add propidium iodide to cells. Cells take up the dye and intercalate with DNA. The amount of stain is proportional to the amount of DNA |
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What are cell cycle regulators?
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cyclins, cdks, cyclin-dependent kinase inhibitors (cki's)
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What regulates cyclins?
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transcription, translation, proteosomal degradation
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What regulates cdk's?
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phosphorylation, dephosphorylation
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What do cki's do?
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Binds cyclin or cyclin/cdk and causes inactivation of cdk activity
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what are the two families of Cki proteins?
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CIP/KIP - bind to cyclin/cdk complex (ternary - binds to something that binds to the cdk)
INK4 - bind cdk - binary (binds directly to the cdk) |
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what is the purpose of cell cycle check points?
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to make sure that damaged chromosomes dont continue on to get replicated and passed onto daughter cells. Also, to make sure that genome is only replicated once per cell cycle.
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What happens if you lose both copies of the Rb tumor suppressor gene?
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uncontrolled cell growth, tumor
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What are the two forms of control at the G1 checkpoint? (to block G1 to S)
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1. Low level of DNA damage: induces low-level p53 expression --> growth arrest --> upregulation of p21 expression --> p21 binds to CDK, blocks G1/S progression
= cytostatic function 2. High level of DNA damage: induces high level p53 expression --> induction of Bax protein --> cell death = intrinsic pathway of apoptosis |
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What is Bax?
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Member of Bcl2 family of proteins
acts at mitochondria to induce cell death by blocking the action of Bcl2, so that the membrane is permeable to cytochrome c (a potent death effector) |
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How is apoptosis triggered?
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Bax or Bak induces the release of death-promoting factors (ie. cytochrome c) . death inducers (like cytochrome c) are stored in intermembrane space of the mitochondria. cytochrome c activates initiator caspase 9.
cytochrome c activates adaptor protein --> assembly of adaptor proteins --> recruitment of procaspase 9 molecules --> apoptosome |
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How is procaspase activated?
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Cleaved (into caspase molecules)
Procaspase = inactive enzyme (proenzyme) |
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What is caspase?
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cystine-aspartase protease
Cleaves other proteins - required for apoptosis. Can cleave and activate other caspases. (so initiator caspases cleave and activate executionor caspases) Caspases also activate the endonucleases that cleave DNA. |
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What is caspase 9?
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Initiator caspase, activated in response to cytochrome c release
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What are the anti and pro-apoptotic members of the Bcl2 family?
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anti: Bcl2, BclXL
pro: Bax, Bak, Bid exist as dimers, which affects function. Ie. Bcl2 + Bax = not pro-apoptotic |
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How do pro-apoptotic proteins work?
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Form channels or pores in the mitochondria that allow cytochrome c release
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What happens in B cell lymphoma?
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Bcl2 driven by a very active promoter (due to chromosomal translocation), which blocks cell death after massive clonal expansion.
Bcl2 = first apoptotic gene implicated in cancer |
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What morphological changes occur in an apoptotic cell?
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- At the very end, you see TB (trypan blue), which can only pass through a compromised cell membrane (so no longer in tact)
cells shrinkage, blebbing, chromatin condensation, DNA degradation, fragmentation of nucleus (then fragmentation of cell..), apoptotic bodies |
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What are apoptotic cells? What happens to them?
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organelles, degraded DNA, other cellular material wrapped in a cellular membrane.
Engulfed by neighboring cells/macrophages |
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What is the signal for macrophages to engulf dying cells?
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Phosphitadylserine (phospholipid) flipped to outer leaflet
(PS can be tagged with annexin V, a Ca-dependent protein which has a high affinity for PS) |
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How can you tell if a cell is dying of apoptosis or necrosis?
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Cell morphology
DNA fragmentation patterns Phosphatidylserine localization to outer leaflet |
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How is chromosomal DNA fragmented/degraded? When does this occur in apoptosis (beginning or end)?
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In a DNA ladder pattern
- this is the final stage of apoptosis |
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Name 3 chemotherapeutic agents (to treat cancer). How do they act?
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etoposide (topoisomerase II) -
camptothecin (topoisomerase I) - natural product drug taxol - induce endonucleolytic DNA cleavage |
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what is cytostatic? cytotoxic?
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Cytostatic - induces growth arrest
Cytotoxic - induces cell death (apoptosis) |
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How can you measure the efficacy of camptothesin, other chemotherepeutic agents, to kill cancer cells?
Can you always use this method to measure apoptotic cell death? |
Flow cytometry shows new population of cells in Sub-G1 - b/c they have less than 2n content of DNA during apoptosis
No - only can be used for intrinsic apoptotic pathway. |
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What is the extrinsic pathway of apoptosis?
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Receptor/ligand interaction can activate caspace 8,10 without cytochrome c release from mitochondria.
caspase 8 activates downstream executioner caspaces (3,6,7), endonucleases, etc. |
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How does the TRAIL induction of death receptors occur?
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- ligand docks on trimerized receptor
- Formation of DISC (Death-inducing signaling complex c) - Induction of FADD (Fas associated death domain) - Release of active caspase 8,10 - Activation of effector caspases 3,6,7 |
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How is Rb regulated?
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Central Control system communicates with the environment via growth factor signaling to regulate Rb
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Where is the Rb restriction point?
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Before S (DNA replication)
*between G1 and S |
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How is Rb inactivated?
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phosphorylated by Cdk
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What happens if a cell does not express Rb?
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uncontrolled cell growth regardless of the nature of the environment (--> mutations)
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What does Cyclin D do?
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Major cyclin controlling progression from G1 to S
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How many D cyclins are in mammals?
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3 (Cyclin D1, D2, D3)
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What does p21 CKI binding result in?
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Blocks CDK activity
Rb remains active |
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What percent of cancers have lost Rb function?
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50%
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What is the initiator caspase?
executioner caspases? |
initiator: Caspase 9, 8, 10
executioner: Caspase 3, 6, 7 |
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What can caspases cleave?
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- endonucleases to activate them
- other caspases - important structural proteins, like lamins |
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How do anti-apoptotic members of the Bcl2 family work?
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bind to pro-apoptotic proteins and prevent them from working
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What does the initiator caspase do?
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Cleave other caspases, which then become executioners which kill the cell
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What happens in necrosis?
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Cell membrane compromised entirely.
Genomic DNA spills out into external millieu (tissue, blood...) Sets up inflammatory response in body |
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What does FLIP do?
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In extrinsic apoptotic pathway...FLIP blocks caspase 8,10 activation (so prevents apoptosis)
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Both the intrinsic and extrinsic apoptotyic pathways have crosstalk through what protein?
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Bid
(can cause release of cytochrome c) |