Chapter 10 Flashcards
Compare and contrast the processes of necrosis and apoptosis
NECROSIS Passive Inflammation No caspases Cell swelling Loss of membrane integrity Swelling of cytoplasm and mitochondria Membrane asymmetry preserved Random digestion of DNA Ends w/ total cell lysis
APOPTOSIS
Active, ATP required Nuclear condensation
No inflammation (cleared by phagocytes)
Caspases activated
Cell shrinkages and breaking into apoptotic bodies
Membrane blebbing, alteration of symmetry
Shrinking of cytoplasm
List 4 changes seen in cells undergoing apoptosis
Membrane blebbing
Cytoplasm shrinkage
Nuclear condensation
Apoptotic bodies (cell frags seen late in apoptosis) - no loss of membrane integrity
Define Anoikis
Apoptosis that is induced in response to inadequate cell-matrix interactions
Overexpression of Bcl-2 in ___ (type of human cancer) led to inhibition of ____ in those cells
Human follicular B cell LSA
Apoptosis
List 4 anti and 4 pro-apoptotic proteins identified in mammalian cells
ANTI-Apoptosis: Bcl-2, Bcl-Xl, Bcl-w, Mcl-1
PRO-Apoptosis: Bax, Bak, Bad, Bik, Bim, Bid
Describe the 2 pathways of apoptosis - include how apoptosis is triggered in these pathways and the intermediate signaling molecules that are involved.
Also, what pro-apoptotic molecule links the two pathways…
EXTRINSIC - death receptor pathway:
Fas/FasL binding activated pro-caspase 8 to caspase 8 which activates caspases 3,6,7 leading to apoptosis
INTRINSIC - mitochondrial pathway
DNA damage stimulates p53 which activates Bax. Bcl-2 normally sits on mitochondrial membrane surface. When Bax is in higher concentration than Bcl-2 in the cell, Bcl-2 no longer plugs holes in the mitochondria so cytochrome C is released from the mitochondria. Cytochrome c binds w/ APAF-1 and caspase 9 to form the apoptosome which activates caspases 3,6,7 and leads to apoptosis.
BID links death receptor pathway to the mitochondrial pathway to amplify the death receptor signaling pathway
IAPs - inhibitors of apoptosis inhibit activation of 3,6,7 by the apoptosome.
SMAC/DIABLO inhibits IAPs
(p197)
What are caspases?
How are they activated?
What are the 2 groups they are divided into based on their function?
Caspases: cysteine proteases in the cytosol
Normally in an inactive form (pro-caspase)
To become active, must be cleaved at specific aspartate residues.
2 groups: initiator (8,9,10) and effector (3,6,7) caspases
p53 is activated by 2 major pathways, what are they?
Mitogenic signals
DNA damage
When p53 is activated, are pro- or anti- apoptotic proteins activated?
PRO apoptotic
T/F - p53 is required for apoptosis to occur?
False. P53 is nb but not required
Name 3 receptors from the death receptor pathway
Fas (aka CD95)
TNFR-1 (tissue necrosis factor receptor 1)
Trail (TNF related apoptosis inducing ligand)
Describe how alterations of cell membrane structure during apoptosis can be used to evaluate for the presence of apoptosis
In normal cells, neutral phospholipids are in the outer membrane; inner membrane is composed of anionic phospholipids such as PHOSPHATIDYLSERINE (PS). When undergoing apoptosis, anionic phospholipids such as PS are exposed on the outer membrane (10.6).
ANNEXIN-V has high affinity for PS and marks cells w/ PS on the outer membrane. Thereby marking cells undergoing apoptosis.
Describe how the propidium iodide stain is used to ID cells undergoing apoptosis
PI difuses into cells w/ compromised cell membranes and binds cellular DNA. T/F they target cells in late apoptosis. This test is usually combines w/ ANNEXIN-V to distinguish early from late apoptotic cells (10.6B)
Describe how cytochrome c IHC can be used to ID cells undergoing apoptosis
If normal cell, cytochrome c has punctate staining in mitochondria. If undergoing apoptosis, staining will be diffuse t/o cell bc it was released from the mitochondria.
Describe the TUNEL assay to assess apoptosis
TUNEL - terminal transferase (TdT) mediated dUTP nick end labeling
TdT catalyzes the addition of a fluorescein labeled dUTP to the ends of double and single stranded DNA in apoptotic cells, the cells are then visualized w/ flow cytometry
