2. Brief glance at the apoptotic process
Role of apoptosis in healthy physiology, esp. in
Immunity
Protection of genome
Disruption of apoptosis & its consequences, viz.
Neurodegeneartive diseases
Cancer
Chronic inflammatory diseases
Potential therapeutic roles
OBJECTIVES
3. 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.
INTRODUCTION
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 for self defense
Examples:
Cells infected with viruses
Cells of the immune system
Cells with DNA damage
Cancer cells
5.
6.
7. 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-α)
Lymphotoxin (TNF-β)
Fas ligand (FasL)
8. Cell death by injury
-Mechanical damage
-Exposure to toxic chemicals
Cell death by suicide
-Internal signals
-External signals
CAUSES OF CELL DEATH:
9. 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)
10. 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
15. Bleb
Blebbing & Apoptotic bodies
The control retained over the cell
membrane & cytoskeleton allows intact
pieces of the cell to separate for
recognition & phagocytosis by MΦs
Apoptotic body
MΦ MΦ
19. 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
20. 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
21. 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
23. Importance of Apoptosis
• Important in normal physiology / development
– Development:
– Immune system maturation
– Morphogenesis
– Neural development
– Adult:
– Immune privilege
– DNA Damage
– Wound repair.
24. Immune system maturation:
Positive selection of thymocytes in the
thymus. Thymic selection involves
thymic stromal cells (epithelial cells,
dendritic cells, and macrophages), and
results in mature T cells that are both
self-MHC restricted and self-tolerant.
25. Immune system maturation:
Negative selection of thymocytes in the
thymus. Thymic selection involves
thymic stromal cells (epithelial cells,
dendritic cells, and macrophages), and
results in mature T cells that are both
self-MHC restricted and self-tolerant.
27. Immune Privilege:
Fas-ligand (FasL; also called CD95L or Apo-1L)
required for tissues to display a privileged status
FasL functions to induce apoptotic cell death in most
cells that express its receptor, Fas.
Fas-bearing cells include cells of the immune
system
Tissues that naturally express FasL kill
infiltrating lymphocytes and inflammatory cells.
28. DNA Damage
Transcriptional Up-Regulation
of Target Genes
p21
(CDK inhibitor)
GADD45
(DNA repair)
BAX
(apoptosis gene)
Ionizing Radiation,
Carcinogens & Mutagens
DNA Damage
p53 activated and binds to DNA
G1 Arrest
Successful repair
Repair fails
APOPTOSIS
Role of apoptosis
In maintaining
Integrity of genomic
DNA in normal cells
29. Disruption of apoptosis
Two major ways:
Inappropriate activation of the
apoptotic process
Immune defect in AIDS
Neurodegenerative diseases.
Inadequate apoptosis
Cancer
Chronic inflammatory conditions
Autoimmune diseases.
30. Immune defect in AIDS
Profound reduction in the population size of CD4
+ T helper cells
Caused by excessive apoptosis
Process includes transfer of regulatory viral gene
products (such as HIV-1 Tat) from HIV infected
cells to bystander T cells
Renders them susceptible to T cell receptor-
induced, CD95-mediated apoptosis.
31. Neurodegenerative Disease
Apoptosis triggered by
amyloid β
neurotoxic abnormal protein structures or aggregates
In adult neurodegenerative diseases including
Alzheimer's
Huntington's chorea
Parkinson's disease,
Amyotrophic lateral sclerosis
Amyloid β can exert neurotoxic effects by
generation of intracellular oxidative stress
increases in calcium ions
Both of these can trigger apoptosis in susceptible
cell types.
32. Cancer
Mutations affect the control mechanisms of
apoptosis and cell survival .
Bcl-2 in follicular lymphoma
increased bcl-2 expression confers resistance to
chemotherapy in ALL and some forms of AML
Bcl-2 blocks the endonucleolytic cleavage of DNA
that is so characteristic of apoptosis.
BCR-ABL in CML
Inappropriately prolongs cell survival by inhibiting
apoptosis
Recent evidence indicates that BCR-ABL can mimic
the modularity signals provided by some cytokines
involved in apoptosis.
33. Chronic Inflammatory conditions:
Intact neutrophils are engulfed by macrophages
at the sites of inflammation.
Morphological changes and a chromatin
fragmentation pattern, characteristic of
apoptosis, within the neutrophils triggers
recognition by the macrophages.
Rheumatoid arthritis may reflect prolonged
survival of leucocytes that are normally
programmed to die by apoptosis.
34. Therapeutic Significance:
Approaches to counter inappropriate apoptosis:
Caspases
are critical to the control of apoptosis
several pharmaceutical companies are developing
potent and specific caspase inhibitors
have shown great promise in murine models of
inappropriate neuronal apoptosis.
The treatment of certain lymphomas by antisense
oligonucleotides to bcl-2
Death-inducing cytokines of the tumour necrosis
factor family, such as TRAIL
35. SUMMARY:
Apoptosis: mechanism & regulation
Importance of apoptosis during early
development
Importance of apoptosis during adulthood
Disruption of apoptotic pathway
Therapeutics
36. References:
Why is apoptosis important to clinicians; Haslett C; BMJ.
2001 June 23; 322(7301): 1499–1500.
Association of Tumor Necrosis Factor-Related Apoptosis
Inducing Ligand with Total and Cardiovascular Mortality
in Older Adults; Stefano V et al; Atherosclerosis. 2011
April ; 215(2): 452–458.
doi:10.1016/j.atherosclerosis.2010.11.004.
Apoptosis: a basic biological phenomenon with wide-
ranging implications in tissue kinetics; Kerr JF, Wyllie
AH, Currie AR; Br J Cancer. Aug;26(4):239-57
Pathologic Basis of Disease; Robbins & Cotran
Immunology, 5th
Ed; Kuby
39. 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
40. 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
41. 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
42. 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
CTL Virally
infected
cell
Fas/ CD95 is the
‘death receptor’
The immunological
synapse holds the cells
much tighter together
than shown here
