Cell Death Flashcards
Generally describe the three methods of Cell Death.
- Apoptosis: programmed cell death, clean, contained.
- Autophagy is a survival pathway where the cell digests itself for nutrients in times of stress. However it can also kill the cell if digest too much
- Necrosis is just murder, its messy and widespread death. In response to an external stimulus.
What are three conditions caused by apoptosis.
Syndactyl (fused fingers)
Lack of normal brain development
Tail Reabsorption.
What are morphological markers of apoptosis.
Blebbing
Plasma membrane in tact
Large, clear vacuoles
Apoptotic bodies (membrane that breaks off, they are vesicles containing nucleic acids) - they will
Nuclear fragmentation
Cell shrinkage.
PS flipping: incubate cells with Annexin V, labels phosphotidyl serines. During apoptosis they will flip towards the extracellular. Then do an antibody that recognizes PS. Assays membrane changes.
TUNEL - it detect strand breaks, high sensitivity, fast, high precision, detergent can make false positives, use radioactively label nucleotides that will insert into the DNA.
DNA laddering: there is a distinctive DNA laddering for DNA degraded by capsase activated DNase (CAD). It cleaves DNA at internucleosomal linker regions resulting in fragments that are multiples of 180.
Immunohistochemistry for caspase: 33kb for inactive, if the 17kb fragment is present that means it active.
Describe the intrinsic apoptosis pathway.
It works through the mitochondria.
DNA damage sensed by p53 which decides on cell death will activate sensors (BH3 proteins- Bid and Bad) which antagonize BCL2. p53 upregulates pro-apoptotic members PUMA and BAX.
BCL2 which blocks binding of Bax, Bak to the mitochondria is removed. Bax and Bak pokes holes into the mitochondria and this releases cytochrome C. Cytochrome C complexes with APAF1 forming apoptosome to activate caspase 9 which goes on to cleave and activate Caspase 3,6,7 which do the killing.
How does p53 monitor cell activity and condition?
p53 is kept in low levels in unstressed cells. Mdm2 will mark p53 for degradation by the proteosome or exporting it to the cytoplasm (as a TF) that makes it useless.
p53 is activated by phosphorylation of its N-terminal domain. Protein kinases that activated are MAPK and checkpoint kinases( ATM, ATR, CHK).
Oncogenes also stimulate p53 activation.
Exposure to Stress will lead to p53 dissociating from the Mdm2.
How do cancers use Bcl-2?
Cancers will perform recombinations, causing the gene for Bcl-2 to be placed next to promoter IgG (for lymphocytes) a very strong promoter. The Bcl-2 balance will be so heavy in favor of Bcl that apoptosis can’t take place. There is uncontrolled division.
How are capsases activated.
How does this lead to DNA fragmentation.
Initiator caspases need to be activated by two proteolytic cleavages.
These capsases leap onto effector caspases 3,6,7 and cleave two sites.
Caspase 3 and 7 cleave a CPAN dimer which goes onto becoming a active DNAse (CAD).
Describe the extrinsic apoptosis pathway.
It is independent of p53 and the mitochondria.
There is a death receptor 4 and 5. When a pro-apoptotic ligand (Apo2L/TRAIL) binds, they activate the intracellular domain which binds FADD (death domains) that ultimately recruit and fuse with procaspases 8 and 10 to form a DISC. (death inducing signal complex)
DISC acts like an enzyme and activates caspase 8,10 which goes on to activate 3,6,7
Describe cross talk between the intrinsic and extrinsic pathways.
They converge at the effector caspases 3,6,7
One example is caspase 8 and 10 can activate BID, BID will tie up BCL2 which allow Bax and Bak to work and cytochrome C to leak out.
Describe autophagy and its four stages
It is usually a survival pathway and is responsible for degrading cellular proteins and organelles. When nutrient levels are low, cells self cannibalize.
Autophagy is the sequestering of organelles into cytoplasmic autophagic vacuoles (autophagosomes) which fuse with lysosomes. On the EM, the cell is full of vacuoles.
- induction: mTor (serine threonine kinase) - under starvation conditions it is inactivated and can’t inhibit autophagy. This allows for expression of autophagy related genes.
- autophagosome formation: these proteins participate in the formation of a two layer membrane surrounding the organelle which will fuse with the lysosome.
- the autophagosome fuses with the lysosome and releases its contents into the lysosome for degradation.
- And everything is broken down into its constituents.
Describe necrosis
It is death by external trauma like toxins, infection, physical trauma.
Necrossi is always detrimental and fatal, and irreversible.
There is cytoplasm spewing every where and there is a huge inflammatory response.
So about p53 and cell death…
phosphorylation of p53 and MDM2 inhibits their association, ARF1?
proteins which regulate release of pro-apoptotic factors contain BH3 domains.
