Apoptosis is a programmed cell death and this can by due to physiological

Apoptosis

1. APOPTOSIS: An overview
Sanjeev Sharma*, Aarti Bhardwaj
$
, Shalini Jain
#
and Hariom Yadav
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*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.

2. INTRODUCTION
Cell death by injury
-Mechanical damage
-Exposure to toxic chemicals
Cell death by suicide
-Internal signals
-External signals

3. 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.

4. 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

5. 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-β)
•Fas ligand (FasL)

6. History of cell death / apoptosis research
1800sNumerous observation of cell death
1908Mechnikov wins Nobel prize (phagocytosis)
1930-40 Studies of metamorphosis
1948-49 Cell death in chick limb & exploration of NGF
1955Beginning of studies of lysomes
1964-66 Necrosis & PCD described
1971Term apoptosis coined
1977Cell 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)

7. Necrosis vs. 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
Necrosis Apoptosis

8. NECROSIS Vs APOPTOSIS
Wilde, 1999

9. STAGES OF APOPTOSIS
Sherman et al., 1997
Induction of apoptosis related genes, signal transduction

10. membrane
blebbing &
changes
mitochondrial
leakage
organelle
reduction
cell
shrinkage
nuclear
fragmentation
chromatin
condensation
APOPTOSIS: Morphology
Hacker., 2000

11. membrane blebbing & changes
mitochondrial leakage
organelle reduction
cell shrinkage
nuclear fragmentation
chromatin condensation
APOPTOSIS: Morphological events

12. Bleb
Blebbing & Apoptotic bodies
The control retained over the cell
membrane & cytoskeleton allows intact
pieces of the cell to separate for
recognition & phagocytosis by Ms
Apoptotic body
M M

13. Caenorhabditis elegans
1090 cells 131 cells apoptosis
ced-1
ced-2
ced-5
ced-6
ced-7
ced-10
ced-3
ced-4
ced-9egl-1
ces-1ces-2
nuc-1
execution
decision
to die
engulfmentdegradation

14. Apoptosis: Pathways
Death
Ligands
Effector
Caspase 3
Death
Receptors
Initiator
Caspase 8
PCD
DNA da
mage
& p53
Mitochondria/
Cytochrome C
Initiator
Caspase 9
“Extrinsic Pathway”
“Intrinsic Pathway”

15. MAJOR PLAYERS IN
APOPTOSIS
•Caspases
•Adaptor proteins
•TNF & TNFR family
•Bcl-2 family

16. Ligand-induced cell death
Ligand Receptor
FasL Fas (CD95)
TNF TNF-R
TRAIL DR4 (Trail-R)

17. Ligand-induced cell death
“The death receptors”
Ligand-induced trimerization
Death Domains
Death Effectors
Induced proximity
of Caspase 8
Activation of
Caspase 8
FasL
Trail
TNF

18. p53
Apoptosis events
Initiator caspases
6, 8, 9,12
Activators of
initiator enzymes
Apoptotic signals
Execution caspases
2, 3, 7
APOPTOSIS: Signaling & Control pathways I
Externally driven
Internally
driven
Cytochrome C
Externally driven
Activation
mitochondrion

19. p53
External
Internal
Apoptosis events
Initiator caspases
6, 8, 9,12
Activators of
initiator enzymes
Apoptotic signals
Execution caspases
2, 3, 7
Inhibitors of
apoptosis
APOPTOSIS: Signaling & Control pathways II
Inhibitors
Externally driven
Internally
driven
Cytochrome C
Externally driven
Survival
factors
Bcl2
Inhibition

20. H
2
O
2
Growth factor
receptors
casp9
Bcl2
PI3K
Akt
BAD
Apaf1
Cyt.C
ATP
The mitochondrial pathway
casp3
casp3
IAPs
Smac/
DIABLO
AIF
Bax
Bax
p53
Fas
Casp8
Bid
Bid
Bid
DNA
damage
Pollack etal., 2001

21. 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)

22. 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

23. 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

24. 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.

27. DNA DAMAGE
p53

28. The bcl-2 family
BH4 BH3 BH1 BH2 TMN C
Receptor domain
phosphorylation
Raf-1
calcineurin
Pore
formation
Membrane
anchor
Ligand
domain
Group I
Group II
Group III
Bcl-2
bax
Bad
bid
bik
Back

29. 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

30. 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

31. Cytolytic lymphocyte/CTL (& natural killer lymphocyte)
presents Fas ligand/CD178 on its surface to tell the infected
cell to die
Apoptosis events
Initiator caspases
Apoptotic signals
Execution caspases
Externally driven
Cytochrome c
Fas ligand
Apoptosis signal to kill infected cells
CTLVirally
infected
cell
Fas/ CD95 is the
‘death receptor’
The immunological
synapse holds the cells
much tighter together
than shown here