Cell Death Flashcards
Intrinsic mechanism of apoptosis initiated by
Irreparable DNA damage
Hypoxia
Increase Ca levels
Severe oxidative stress
Steps in apoptosis
Cell shrinkage Chromatin condensation/ margination Membrane blebbing Nuclear collapse Apoptotic body formation Lysis of apoptosis bodies
Intrinsic pathway - apoptosis
Increase p53, increase BAX.
Permeable mitochondria membrane.
Release of SMAC/ DIABLO -> inhibits inhibitors of apoptosis IAPs
Release of cytochrome C, binds apoptosis protease activity factor Apaf 1. Forms apoptosome which activates caspase 9.
Activation of effector caspase a 3, 6, 7.
Extrinsic pathway - apoptosis
Proapoptotic ligand a bind receptor (TNF/Fas)
Recruitment of death domains (TRADD/FADD)
Procaspase 8 (initiator caspase) binds and forms DISC - death inducing signaling complex.
Procaspases brought into close proximity, auto catalytic processing.
Activates effector caspase a 3,6,7.
What do caspases do?
Activate endonucleases and proteases
3,8 regulate phosphatidyl serine expression
3,6,7 cleave substrates.
Biochemical features of apoptosis
Protein cleavage Protein cross linking DNA breakdown Phosphatidyl serine expression Caspases
Assays for apoptosis
DNA fragmentation - TUNEL assay
- caspase activated DNase cut internucleosomal sites
Caspases detection - ELISA
- Western BLOT
Alteration in membrane - annexing V assay
Mitochondrial assay - laser scanning confocal microscopy LSCM
Types of necrosis
Liquifactive Coagulative Haemorrhagic Toxin induced Viral Caseous
Necrosis - changes in cell morphology
Increase size, organelles swell, plasma membrane rupture, loss of cell contents.
Nuclear changes in necrosis
Chromatin clumping, karyohexis, pyknosis, karyolysis.
Triggers of autophagy
Protein aggregation Organelle damage Nutrient depletion Growth factor deprivation Microbial infection
Three types of autophagy
Microautophagy
Chaperone mediated autophagy
Macroautophagy
Process of microautophagy
Cytoplasmic contents enter directly into lysosomes. Degraded by hydrolases
Process of chaperone mediated autophagy
Hsc70 recognizes KFERQ.
Co chaperones unfold protein.
Lamp2 translocates protein into lysosomes.
Degraded by hydrolases.
Process of macroautophagy
Engulfment of components by phagophore.
Phagophore extends creating double membrane vehicle = autophagosome.
Fuses with lysosome. Inner membrane and protein content hydrolysed = autolysosome.
Pathways regulating autophagy
mTOR dependent
mTOR independent
Uses of autophagy
Adaption for longer periods of starvation
Elimination of cytoplasmic contents
Simple pathway from cytosol to lysosomes
Sequestration with degradation eg ER stress
Hayflick limit
Finite replicative potential of normal somatic cells in culture
Use of senescence
Prevent genomic instability
Prevent cancer
Two types of senescence
Replicative senescence
Accelerated senescence
Bypassing senescence in culture
Grow on fibroblast feeding layer
Ectopic expression of hTERT
Suppression of p53
Simian virus 40 infection
Markers of senescence
Fail to proliferate in response to growth factors
Not sensitive to apoptotic stimuli eg serum withdrawal
P53, p16, RB levels
Morphology - large, thin, stress fibers
Assay for senescence
B-gal positive at pH 6
Quiescence
Reversibly growth arrested cells
Markers of quiescence
Sensitive to apoptotic stimuli
GFs cause proliferation
B-gal neg
Morphology - cobblestone.