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39 Cards in this Set
- Front
- Back
What is flow cytometry?
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Technique used to determine the number of cells in G1, S, or G2-M. Add propidium iodine - they take up dye that goes to nucleus and intercalates into DNA. Amount of stain directly proportional to amount of DNA.
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What is the mammalian cell cycle basic regulatory apparatus?
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cyclins, cdks, and ckis (cyclin dependent kinase inhibitors)
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What controls progression between S and M phase?
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It is a conserved regulatory apparatus - links cell cycle with extracellular signals that control proliferation
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What regulates cyclin?
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Regulated by transcription, translation, and proteasomal degradation
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What regulates Cdk activity?
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phosphorylation/dephosphorylation
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What are CKIs?
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protein that binds to either cyclin/cdk complex or cdk and inactivates cdk activity
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What is the major G1 cdk?
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Cyclin D with Cdk4 and Cdk6 partners. Cyclin D binds to all G1 and G1/S Cdks. It is upregulated by growth factor stimulation of transcription
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What does Rb control?
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The restriction point. If Rb is non-functional, will have uncontrolled growth.
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How can growth arrest be induced?
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G1 growth arrest - increased destruction of cyclin D, increased phosphatase action to dephosphorylate kinase CDK4 or CDK6
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What are the 2 families of CKI proteins?
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CIP/KIP - Bind to cyclin/CDK complex
INK4 - find to the CDK Both occur at G1/S checkpoint |
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If CKI is bound to a complex, is it active or inactive?
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INACTIVE! blocks the ability of the complex to phosphorylate Rb.
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Why is there a cell cycle check point?
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TO insure that incomplete or damaged chromosomes are not replicated. Ensure genome is replicated only once per cell cycle.
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Restriction point
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Controlled by Rb. Only one copy is typically sufficient. Rb is compromised in many tumors.
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What are the 4 other checkpoints (not one with Rb)?
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-G1/S - DNA damage
-G2/M - Is cell big enough, DNA replicated, DNA damaged? -Mitotic Spindle - Are all chromosomes aligned? - S phase - is all DNA replicated |
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Role of p53 with low level DNA damage
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Low level DNA damage - induces p53 and growth arrest. p53 induces p21, p21 binds to CDK and blocks G1/S progression.
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Role of p53 with high level of DNA damage
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Induces high levels of p53 and Bax. (Bax is a Bcl2 protein). Bax acts on mitochondria by blocking Bcl-2, making mitochondrial wall permeable - releases cytochrome C - promotes intrinsic pathway of cell death.
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What are two pro-apoptotic members of the Bcl2 family?
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Bax and Bak
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Intrinsic Pathway of Apoptosis
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Induced by p53 and other effector proteins
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Where is cytochrome C located?
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In the mitochondrial intermembraneous space
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What does cytochrome C active?
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Cytochrome C activated an Adaptor protein. It assembles and recruits procaspase 9 to form an apoptosome
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How is procaspase activated?
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Caspases exist as proenzymes. Proteases (cysteine/aspartate) are involved. They activate endonucleases that cleave DNA.
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What are two anti-apoptotic members of the Bcl2 family?
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Bcl2, BclXL. Pro-apoptotic are Bax, Bak, Bid
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Do cancer cells have to have a chromosomal translocation event to upregulate Bcl2 levels?
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No
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What are characteristics of an apoptotic cell?
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Cell membrane is intact until the end. (Trypan Blue dye can show this - it can only pass through a compromised membrane) Has shrinkage, blebbing, chromatin condensation, DNA degradation, nuclear fragmentation, apoptotic bodies
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What is an apoptotic body?
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cell membrane enveloped cellular organelles, degraded DNA, and other material. Engulfed by cells or macrophages
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phosphotidylserine in dying cell
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This does not set up an inflammatory reaction. PS is flipped to the outer leaflet in a dying cell, annexin V binds to dying cell.
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DNA degradation during apoptosis
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Chromosomal DNA is fragmented due to endonuclease cleavage - cleaves at linker regions. Flow cytometry shows a portion in subG1 area. Less than 2n
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Extrinisic Pathway of apoptosis
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Happens WITHOUT cytochrome C. Receptor/ligand interaction activates caspases 8 &/or 10 - activates executioner caspases 3, 6, 7 and endonucleases
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How are the intrinisic and extrinisic apoptosis pathways connected?
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They are connected through Bid. p53 can activate caspase-8, which activates Bid, t-Bid activates cytochrome C.
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What does the loss of p53 cause?
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leads to uncontrolled growth and genomic instability where genes may be amplified. Both alleles of p53 must be mutated or deleted
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What are some mechanisms for Multidrug resistant cancer cells?
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Loss of p53
Loss of Rb cyclin D up-regulation growth factor up-regulation increased GF receptors Bcl2 upregulation |
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What regulates Rb activity?
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cyclinD/CDK
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What is the clinical application of flow cytometry?
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determine efficacy of cancer drugs. See growth arrested cells. Proliferating population
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What are common initiator/executioner caspases?
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Initiator - 8,9,10
Executioner - 3, 6, 7 |
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What does caspase 3 target?
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activates endonuclease
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What is the target for caspase 6?
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cleaves Lamin A
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What is the target for caspse 7?
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cleaves poly ADP ribose polymerase
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What is necrosis?
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When a cell membrane is compromised early. Genomic DNA and contents of cell spill out. Causes inflammatory response
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What is camptothecin?
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It inhibits topoisomerase I
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