APOPTOSIS: An overview

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APOPTOSIS An overview

• Sanjeev Sharma, Aarti Bhardwaj, Shalini Jain
  and Hariom Yadav
• Animal Genetics and Breeding Division, Animal
  Biochemistry Division, National Dairy Research
  Institute, Karnal-132001, Haryana, India
• College of Applied Education and Health
  Sciences, Meerut, U.P.

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INTRODUCTION

• Cell death by injury
• -Mechanical damage
• -Exposure to toxic chemicals
• Cell death by suicide
• -Internal signals
• -External signals

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Conted..

• Apoptosis or programmed cell death, is carefully
  coordinated collapse of cell, protein degradation
  , DNA fragmentation followed by rapid engulfment
  of corpses by neighbouring cells.
  (Tommi, 2002)
• Essential part of life for every multicellular
  organism from worms to humans. (Faddy et
  al.,1992)
• Apoptosis plays a major role from embryonic
  development to senescence.

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Why should a cell commit suicide?

• Apoptosis is needed for proper development
• Examples
• The resorption of the tadpole tail
• The formation of the fingers and toes of the
  fetus
• The sloughing off of the inner lining of the
  uterus
• The formation of the proper connections between
  neurons in the brain
• Apoptosis is needed to destroy cells
• Examples
• Cells infected with viruses
• Cells of the immune system
• Cells with DNA damage
• Cancer cells

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What makes a cell decide to commit suicide?

• Withdrawal of positive signals
• examples
• growth factors for neurons
• Interleukin-2 (IL-2)
• Receipt of negative signals
• examples
• increased levels of oxidants within the cell
• damage to DNA by oxidants
• death activators
• Tumor necrosis factor alpha (TNF-?)
• Lymphotoxin (TNF-ß)
• Fas ligand (FasL)

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History of cell death / apoptosis research

• 1800s Numerous observation of cell death
• 1908 Mechnikov wins Nobel prize (phagocytosis)
• 1930-40 Studies of metamorphosis
• 1948-49 Cell death in chick limb exploration
  of NGF
• 1955 Beginning of studies of lysomes
• 1964-66 Necrosis PCD described
• 1971 Term apoptosis coined
• 1977 Cell death genes in C. elegans
• 1980-82 DNA ladder observed ced-3 identified
• 1989-91 Apoptosis genes identified, including
  bcl-2, fas/apo1 p53, ced-3 sequenced
• (Richerd et.al., 2001)

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Necrosis

• When the cell membrane is damaged
• Necrosis occurs when cells are exposed to extreme
  variance from physiological conditions
• (e.g., hypothermia, hypoxia) which may result in
  damage to the plasma membrane.
• Under physiological conditions direct damage to
  the plasma membrane is evoked by
• agents like complement and lytic viruses.

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Apoptosis

• Apoptosis is a mode of cell death that occurs
  under normal physiological with no damage to cell
  membrane
• conditions and the cell is an active participant
  in its own demise (cellular suicide). It is
• most often found during normal cell turnover and
  tissue homeostasis, embryogenesis,
• induction and maintenance of immune tolerance,
  development of the nervous system
• endocrine-dependent tissue atrophy.

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Necrosis vs. Apoptosis
Necrosis
Apoptosis

• Cellular condensation
• Membranes remain intact
• Requires ATP
• Cell is phagocytosed, no tissue reaction
• Ladder-like DNA fragmentation
• In vivo, individual cells appear affected

• Cellular swelling
• Membranes are broken
• ATP is depleted
• Cell lyses, eliciting an inflammatory reaction
• DNA fragmentation is random, or smeared
• In vivo, whole areas of the tissue are affected

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NECROSIS Vs APOPTOSIS



Wilde, 1999
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STAGES OF APOPTOSIS
Induction of apoptosis related genes, signal
transduction
Sherman et al., 1997
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APOPTOSIS Morphology
organelle reduction
membrane blebbing changes
cell shrinkage
mitochondrial leakage
nuclear fragmentation
chromatin condensation
Hacker., 2000
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Caenorhabditis elegans
1090 cells
131 cells
apoptosis
decision to die
engulfment
degradation
execution
ced-3 ced-4
ced-1 ced-2 ced-5 ced-6 ced-7 ced-10
nuc-1
ced-9
egl-1
ces-1
ces-2
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Apoptosis Pathways
Extrinsic Pathway
Death Ligands
Intrinsic Pathway
DNA damage p53
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MAJOR PLAYERS IN APOPTOSIS

• Caspases
• Adaptor proteins
• TNF TNFR family
• Bcl-2 family

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Ligand-induced cell death

• Ligand Receptor
• FasL Fas (CD95)
• TNF TNF-R
• TRAIL DR4 (Trail-R)

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Ligand-induced cell death
The death receptors
Ligand-induced trimerization
FasL Trail TNF
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APOPTOSIS Signaling Control pathways I
Externally driven
Externally driven
Apoptotic signals
Activators of initiator enzymes
p53
Cytochrome C
Internally driven
Initiator caspases 6, 8, 9,12
mitochondrion
Execution caspases 2, 3, 7
Apoptosis events
Activation
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APOPTOSIS Signaling Control pathways II
Externally driven
Externally driven
Inhibitors
Apoptotic signals
Activators of initiator enzymes
p53
Cytochrome C
Internally driven
Bcl2
Survival factors
External
Initiator caspases 6, 8, 9,12
Internal
Execution caspases 2, 3, 7
Inhibitors of apoptosis
Apoptosis events
Inhibition
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The mitochondrial pathway
Growth factor receptors
Fas
DNA damage
Casp8
PI3K
Akt
p53
Bid
casp3
BAD
Bid
Bax
IAPs
casp9
Bid
Apaf1
Bcl2
ATP
casp3
Bax
Cyt.C
Smac/ DIABLO
H2O2
AIF
Pollack etal., 2001
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REGULATION OF APOPTOSIS

• Stimuli? apoptosis ?selection of targets
  (Rich et al., 2000)
• Apoptosis by conflicting signals that scramble
  the
• normal status of cell (Canlon Raff, 1999)
• Apoptotic stimuli?cytokines, death factors (FasL)
  (Tabibzadeh et al., 1999)
• DNA breaks ? p53 is activated ?arrest cell cycle
  or
• activate self destruction (Blaint Vousden,
  2001)

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Importance of Apoptosis

• Important in normal physiology / development
• Development Immune systems maturation,
  Morphogenesis, Neural development
• Adult Immune privilege, DNA Damage and wound
  repair.
• Excess apoptosis
• Neurodegenerative diseases
• Deficient apoptosis
• Cancer
• Autoimmunity

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FUTURE PERSPECTIVES

• The biological roles of newly identified death
  receptors and ligands need to be studied
• Need to know whether defects in these ligands
  and receptors contribute to disease

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CONCLUSION

• an important process of cell death
• can be initiated extrinsically through death
  ligands
• (e.g. TRAIL, FasL) activating initiator
  caspase 8 through induced proximity.
• can be initiated intrinsically through DNA damage
  (via cytochrome c) activating initiator caspase
  9 through oligomerization.
• Initiator caspases 8 and 9 cleave and activate
• effector caspase 3, which leads to cell death.

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Programed Cell Death helps maintain Health
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DNA DAMAGE
p53
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The bcl-2 family
Receptor domain
Raf-1 calcineurin
Membrane anchor
Ligand domain
Pore formation
phosphorylation
Back
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P53 Apoptosis
p53 first arrests cell growth between G1 ? S
This allows for DNA repair during delay
If the damage is too extensive then p53
induces gene activation leading to apoptosis
(programmed cell death)
BACK
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3 mechanisms of caspase activation
a. Proteolytic cleavage e.g. pro-caspase 3
b. Induced proximity, e.g. pro-caspase 8
c. Oligomerization, e.g. cyt c, Apaf-1 caspase
9
Back
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Apoptosis signal to kill infected cells
Cytolytic lymphocyte/CTL ( natural killer
lymphocyte) presents Fas ligand/CD178 on its
surface to tell the infected cell to die
Fas ligand
CTL
Virally infected cell
Externally driven
Apoptotic signals
Cytochrome c
Initiator caspases
The immunological synapse holds the cells much
tighter together than shown here
Execution caspases
Apoptosis events
Fas/ CD95 is the death receptor