Apoptosis Flashcards
Apoptosis is defined as:
- programmed cell death
- death by suicide
- integral to tissue development
Autophagy is defined as:
- a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery
- death by self-cannibalism
Necrosis is defined as:
- the premature death of cells by external factors
- death by accident or murder
Four functions of apoptosis:
-
Development
- tissue-sculpting
-
Immune System
- eliminating used T and B cells after infection
-
Tissue homeostasis
- balance between cell proliferation and death
-
Cancer prevention
- eliminating cells that have been damaged by UV, radiation, chemical toxins, or viral infection
Syndactyly:
- a condition wherein two or more digits are fused together.
- due to apoptosis misregulation
What is the major distinction between necrosis and apoptosis?
- the cell membrane remains intact during apoptosis - leads to NO immune response.
- it bursts in necrosis, leading to an immune response.
Cell shrinkage and membrane blebbing during apoptosis is due to:
actin cytoskeleton degradation
8 Morphological Markers of Apoptosis:
- Electron-dense nucleus (early phase).
- Nuclear fragmentation.
- Intact cell membranes.
- Disorganized cytoplasmic organelles.
- Large, clear vacuoles.
- Blebs at the surface.
- Loss of cell-cell adhesion.
- Apoptotic bodies.
What is this an image of?
Apoptosis
Cell shrinkage.
What is this an image of?
Apoptosis
Membrane Blebbing
What is this an image of?
Apoptosis
Chromatin Condensation
What is this an image of?
Apoptosis
Apoptotic Bodies
What are the four biochemical markers of apoptosis?
- phosphatidyl-serine flipping (Annexin V)
- increase in DNA nicks (TUNEL)
- DNA laddering (PCR)
- caspase activation (PCR)
Phosphatidyl-serine (PS) flipping:
- a biochemical marker of apoptosis
- phosphatidyl-serine is a phospholipid normally on the cytoplasmic side of the membrane, but flips to the extracellular side during apoptosis.
Phosphatidyl-serine (PS) flipping can be visualized through what method?
- annexin V antibody coupled with GTP
- can use flow cytometry to quantify amount of apoptosis.
Reasoning behind TUNEL:
(Terminal Transferase dUTP Nick-End Labeling)
- DNA in an apoptotic cell has more nicks in it.
- label the nicks with dUTP with a flourescent protein on it.
TUNEL advantages and disadvantages:
(Terminal Transferase dUTP Nick-End Labeling)
- fast, sensitive, reproducible.
- Disadvantages:
- # of breaks necessary for detection unknown.
- necrotic cells cen generate false +.
- detergent used may make cells fragile.
DNA Laddering:
- apoptosis marker.
- distinctive feature of DNA degraded by caspase-activated DNase (CAD).
- CAD cleaves genomic DNA at internucleosomal linker regions, resulting in DNA fragments that are multiples of 180–185 base-pairs in length.
What band on PCR comes up during apoptosis due to Caspase cleavage/activation?
- Cleaved caspase produces a band at 17kDa on PCR; APOPTOSIS.
- Uncleaved caspase produces a band at 33kDa on PCR; NORMAL.
- Lanes 2, 3, and 4 are dying cells.
In order to become active, caspases must be:
- cleaved.
- this process of cleavage is reversible if the apoptotic-causing agent is removed.
What are the two major apoptotic pathways?
- cell intrinsic pathway
- works through mitochondria
- cell extrinsic pathway
- works through “death receptors” on the cell surface that bind pro-apoptotic ligands
The two major apoptotic pathways of the cell converge on what level?
the level of the caspases
Caspases are:
- “molecular hitmen”
- enzymes that give rise to all of the morphological and biochemical changes arising from apoptosis.
- Must be cleaved to become active.
What are the two types of apoptotic caspases?
- initiator (apical) caspases (2,8,9, and 10)
- effector (executioner) caspases (3,6, and 7)
What # caspases are the initiator (apical) caspases?
caspases 2, 8, 9, and 10
- give the order for cell death
What # caspases are the effector (executioner) caspases?
caspases 3, 6, and 7
Caspases are regulated at what level?
- post-translationally
- caspases are always present in the cell in an inactive state.
- can become rapidly activated if needed.
The caspase cascade:
- pro-apoptotic stimulus (i.e. p53).
- intiator caspases cleaved and activated.
- initiator caspases cleave and activate effector caspases.
- apoptosis occurs.
How many proteolytic cleavages are necessary for initator caspase activation?
- two
Steps in how caspase activation results in DNA fragmentation:
- caspase 3 and 7 binds to DFF45/DFF40 dimer.
- DFF45 cleaved and DFF40 released.
- DFF40 oligomerizes into an active DNAase and cleaves DNA.
- leads to DNA ladder on PCR.
The BCL2 superfamily of proteins regulates:
- the integrity of the mitochondrial membrane
- contains pro-apoptotic and anti-apoptotic proteins
What occurs when the integrity of the mitochondrial membrane is compromised?
- it leaks out cytochrome C.
- Cytochrome C interacts with APAF1 to form the apoptosome which will activate the initiator caspases.
- Effector caspases then activated and apoptosis occurs.
Steps in DNA-damage to apoptosis:
- p53 senses DNA damage.
- BH3 sensors BID/BAD/PUMA activated.
- BAX and BAK of BCL2 family activated.
- BAX and BAK compromise integrity of mitochondrial wall. Cytochrome C leaks out.
- Cytochrome C interacts with APAF1 to form the apoptosome.
- Apoptosome activates intiator caspase 9.
- Initiator caspases activate effector caspases 3, 6, and 7.
- Apoptosis occurs.
What proteins in the BCL2 superfamily are anti-apoptotic and maintain integrity of the mitochonrial membrane?
BCL2, BCL-XL, MCL1
- bind to mitochondrial membrane
- no leakage of cytochrome C
What proteins in the BCL2 superfamily are pro-apoptotic and compromise integrity of the mitochonrial membrane?
BAX and BAK
p53 is:
- a transcription factor that regulates the cell cycle and apoptosis
p53 mechanisms of anticancer function (3):
- can activate DNA repair proteins when DNA has sustained damage.
- can arrest growth by holding the cell cycle at the G1/S regulation point.
- can initiate apoptosis if DNA damage proves to be irreparable.
p53 levels in unstressed cells:
- low levels due to continuous degradation
How is p53 kept at low levels in unstressed cells?
- Mdm2 acts as ubiquitin ligase and covalently attaches ubiquitin to p53 and thus marks p53 for degradation by the proteasome.
- Mdm2 also transports p53 from the nucleus to the cytosol.
What protein ubiquitinates p53?
Mdm2
What is the critical event in the activation of p53?
- phosphorylation of the p53 N-terminal domain.
What enzymes can activate p53 through phosphorylating its N-terminal domain?
- Members of the MAPK family
- JNK1-3, ERK1-2, p38 MAPK
- Checkpoint kinases
- ATR, ATM, CHK1 and CHK2, DNA-PK, CAK, TP53RK
- Oncogenes, mediated by the protein p14ARF.
What pro-apoptotic molecules are normally found in the cytosol, translocate to the mitochondrial membrane, and stimulate the formation of pores allowing cytochrome C to leak out?
BAD, BAX, BID
BCL-2 protein:
anti-apoptotic
- binds BAX/BAK and prevents mitochondrial pore formation.
BID and BAD proteins:
promote apoptosis
- tie up BCL-2 and free BAX/BAK.
- BID may also interact with BAX/BAK to open mitochondrial pores.
BAX and BAK proteins:
promote apoptosis
- promote mitochondrial pore formation and release of cytochrome c when APAF1 initiates apoptosis
Follicular lymphoma is due to:
- chromosome translocation that greatly increases the expression of BCL-2.
- cell do not undergo apoptosis, cancer occurs
Conventional anticancer therapies, such as chemotherapy and radiotherapy, activate the intrinsic apoptosis pathway via:
p53
Steps in the Extrinsic Apoptosis Pathway:
- Apo2L/TRAIL ligand binds to pro-apoptotic receptors DR4 and DR5.
- Activated DR4 and DR5 recruit fas-associated death domain (FADD). Death-inducing signaling complex (DISC) forms.
- FADD recruits initiator caspase 8 and/or 10 to the DISC.
- DISC undergoes autocatalysis, activating caspase 8 and 10.
- Activated caspases 8 and 10 are released into the cytoplasm and activate effector capsizes 3, 6, and 7.
- Apoptosis occurs.
What ligand binds to the pro-apoptotic receptors DR4 and DR5 in the extrinsic apoptosis pathway?
Apo2L/TRAIL
Where do the extrinsic and intrinsic apoptosis pathways converge?
- initiator caspases 8 and 10
- BID
- BID is activated by initiator caspases 8 and 10.
- BID ties up BCL-2 and free BAX/BAK, which are all in the intrinsic apoptosis pathway.
Autophagy is responsible for:
- degrading cellular proteins and organelles and recycling them.
- it is a survival pathway.
When is autophagy activated?
- response to cellular starvation.
- when nutrient levels are low, cells self-cannibalize and recycle proteins for metabolism.
- housekeeping process.
- long-lived proteins and organelles are recycled.
- Normal cellular processes.
The four stages of autophagy:
- Induction.
- Autophagosome formation.
- Autophagosome lysosome fusion.
- Autophagosome breakdown.
What protein regulates autophagy under nutrient-rich and starvation conditions?
- mTOR
- inhibits autophagy in nutrient-rich conditions
- in starvation, mTOR is inhibited, which leads to autophagy activation
Many of the autophagy genes that are upregulated in response to mTor deactivation participate in what process?
Autophagosome Formation
- formation of a membrane around a targeted portion of the cell.
What is autophagosome formation?
- the formation of a membrane around a targeted portion of the cell.
Are the effects of necrosis reversible?
No.
Necrosis is caused by:
- factors external to the cell or tissue, such as infection, toxins, or trauma.
Five types of necrosis:
- Coagulation necrosis
- ischemia
- Liquefactive necrosis
- cell destruction leading to escape of hydrolases
- Enzymatic fat necrosis
- escape of lipases
- Caseous necrosis
- bacterial liquefaction
- Gangrenous necrosis
- ischemic + bacterial liquefaction
