Apoptosis

1. APOPTOSIS
Dr.Dinesh T
Junior resident
Department of Physiology
JIPMER
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2. Discussion headings
•Introduction
•Etiopathogenesis
•Morphological, Biochemical changes
•Mechanism – Intrinsic & Extrinsic pathway
•Disorders of apoptosis
•Conclusion
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3. Introduction
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4. Apoptosis - Definition
• A pathway of cell death induced by a tightly
regulated suicidal program, in which the
cells destined to die activate enzymes that
degrade cells own nuclear DNA and nuclear,
cytoplasmic proteins.
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5. Kerr Wyllie and Currie paper, British Journal of Cancer, 1972
Aug;26(4):239-57
"We are most grateful to Professor James Cormack of
the Department of Greek, University of Aberdeen, for
suggesting this term. The word "apoptosis" ( πόπτωσισ)
ἁ
is used in Greek to describe the "dropping off" or "falling
off" of petals from flowers, or leaves from trees. To show
the derivation clearly, we propose that the stress should
be on the penultimate syllable, the second half of the
word being pronounced like "ptosis" (with the "p" silent),
which comes from the same root "to fall", and is already
used to describe the drooping of the upper eyelid
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6. Historical aspects
•German scientist Carl Vogt - Principle of apoptosis
(1842).
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7. •Walther Flemming – Process of programmed
cell death (1845).
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8. •John Foxton Ross Kerr – Distinguish
apoptosis from traumatic cell death
(1962).
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9. Nobel prize in 2002 – Sydney Brenner , Horvitz,
John Buston.
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10. Cell death mechanisms
Death by suicide Death by injury
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11. APOPTOSIS NECROSIS
NATURAL YES NO
EFFECTS BENEFICIAL DETRIMENTAL
Physiological or
pathological
Always pathological
Single cells Sheets of cells
Energy dependent Energy independent
Cell shrinkage Cell swelling
Membrane integrity
maintained
Membrane integrity lost
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12. APOPTOSIS NECROSIS
Role for mitochondria and cytochrome CNo role for mitochondria
No leak of lysosomal enzymes Leak of lysosomal enzymes
Characteristic nuclear changes Nuclei lost
Apoptotic bodies form Do not form
DNA cleavage No DNA cleavage
Activation of specific proteases No activation
Regulatable process Not regulated
Evolutionarily conserved Not conserved
Dead cells ingested by neighboring cellsDead cells ingested by neutrophils and
macrophages
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13. Significance of apoptosis
•During development many cells are produced in excess which eventually
undergo programmed cell death and thereby contribute to sculpturing many
organs and tissues [Meier, 2000]
•In human body about one lakh cells are produced every second by mitosis
and a similar number die by apoptosis (Vaux and Korsmayer ,1999, cell)
•Between 50 and 70 billion cells die each day due to apoptosis in the
average human adult. For an average child between the ages of 8 and 14,
approximately 20 billion to 30 billion cells die a day. ( Karam, Jose A. (2009).
Apoptosis in Carcinogenesis and Chemotherapy. Netherlands: Springer. ISBN
978-1-4020-9597-9)
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14. Etiopathogenesis
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15. Why should a cell commit suicide?
•1. Programmed cell death is as needed for proper normal
development as mitosis is.
Examples:
oThe resorption of the tadpole tail in frog .
oThe formation of the fingers and toes of the fetus requires the
removal, by apoptosis.
oThe sloughing off of the endometrium at the start of menstruation.
oThe formation of the proper connections (synapses) between
neurons in the brain.
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16. •2. Programmed cell death is needed to destroy cells that
represent a threat to the integrity of the organism.
•Examples:
oCells infected with viruses
oCells of the immune system
oCells with DNA damage
oCancer cells (Uncontrolled proliferated cells)
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17. Apoptosis in physiologic situations
o Programmed destruction during embryogenesis
o Involution of hormone dependent tissues
o Cell loss in proliferating cell populations
o Elimination of harmful self- reactive lymphocytes
o Death of host cells
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18. Apoptosis in bud
formation during
which many
interdigital cells
die. They are stained
black by a TUNEL
method

Incomplete differentiation
in two toes due to lack of
apoptosis
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19. Apoptosis: in embryogenesis
Morphogenesis (eliminates excess cells):
Selection (eliminates non-functional cells):
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20. Apoptosis: in embryogenesis
Immunity (eliminates dangerous cells):
Self antigen
recognizing cell
Organ size (eliminates excess cells):
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21. Apoptosis: importantance in adults
Tissue remodeling (eliminates cells no longer needed):
Virgin mammary gland Late pregnancy, lactation
Involution
(non-pregnant, non-lactating)
Apoptosis
Apoptosis
- Testosterone
Prostate gland
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22. Apoptosis: importantance in adults
Tissue remodeling (eliminates cells no longer needed):
Resting lymphocytes + antigen (e.g. infection) - antigen (e.g. recovery)
Apoptosis
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23. Apoptosis in pathological conditions

- DNA damage
- Accumulation of misfolded proteins
- Cell death in certain infections
- Pathological atrophy in parenchymal organs
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24. Cells of the immune system
•CTLs induce apoptosis in each other and even in
themselves.
•Defects in the apoptotic machinery is associated with
autoimmune diseases such as lupus erythematosus and
rheumatoid arthritis.
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25. Cells infected with viruses
•One of the methods by which cytotoxic T lymphocytes
(CTLs) kill virus-infected cells is by inducing apoptosis
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26. Cells with DNA damage
•Damage to its genome
can cause a cell
»to disrupt
proper
embryonic
development
leading to birth
defects
»to become
cancerous.
•Cells respond to DNA
damage by increasing
their production of p53.
p53 is a potent inducer
of apoptosis.
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27. –Cancer cells
•Radiation and chemicals used in cancer therapy induce apoptosis
in some types of cancer cells.
Fig. 1: SC-1 induced apoptosis in stomach carcinoma cells
Left: Before induction
Middle: 24h after induction
Right: 48h after induction sclero dinesh

28. Morphological & Biochemical changes
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29. Classic changes
Cell shrinkage
 Nuclear fragmentation
 Chromatin condensation
Chromosomal DNA fragmentation
Formation of cytoplasmic blebs& apoptotic bodies
Phagocytosis
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30. Histology
Apoptotic bodies
•Round oval mass
of intensely
eosinophillic
cytoplasm
•Fragments of
dense nuclear
chromatin
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31. Bio chemical changes
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32. Mechanisms of apoptosis
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35. Caspases
•Caspase are Cysteine- Aspartic acid specific proteases that mediates
the events that are associated with programmed cell death.
•Their catalytical activity depends on a critical cysteine-residue within a
highly conserved active-site pentapeptide QACRG,
•Caspases specifically cleave their substrates after Asp residues.
• Caspase- 8, Caspase- 9
•acts as an initiator of the caspase activation cascade.
•Caspase-3
•key effector in the apoptosis pathway, amplifying the signal from
initiator caspases and signifying full commitment to cellular
disassembly.
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36. Initiation
•Absence of stimuli - hormones, growth factors
•Activation of receptors – TNF family
•Heat ,radiation, chemicals
•Genetically programmed events
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37. STAGES OF CLASSIC APOPTOSIS
Healthy cell
DEATH SIGNAL / STIMULI (extrinsic or intrinsic)
Commitment to die (reversible)
EXECUTION (irreversible)
Dead cell (condensed, crosslinked)
ENGULFMENT (macrophages, neighboring cells)
DEGRADATION
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38. Receptor pathway (physiological):
Death receptors:
(FAS, TNF-R, etc)
FAS ligand TNF
Death
domains
Adaptor proteins
Pro-caspase 8 (inactive) Caspase 8 (active)
Pro-execution caspase (inactive)
Execution caspase (active)
DeathMITOCHONDRIA
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39. Apoptosis triggered by external signals: the extrinsic or
death receptor pathway
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40. Regulation of apoptosis
•Regulatory proteins – BCL -2, equivalent to
CED -9
•Apoptosis depends on binding of BCL -2 with
pro apoptotic and anti apoptotic proteins.
•Situated in the outer mitochondrial
membrane.
•Apaf -1 equivalent to CED -4.
•Tp 53, caspases, BAX, viruses such as adeno,
papilloma , hepatitis B.
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41. Intrinsic pathway (damage):
Mitochondria
Cytochrome c release
Pro-caspase 9 cleavage
Pro-execution caspase (3) cleavage
Caspase (3) cleavage of cellular proteins,
nuclease activation,
etc.
Death
BAX
BAK
BOK
BCL-Xs
BAD
BID
B IK
BIM
NIP3
BNIP3
BCL-2
BCL-XL
BCL-W
MCL1
BFL1
DIVA
NR-13
Several
viral
proteins
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44. Intracellular signals
Oxidative damage from free radicals, Radiation, Virus infection, Nutrient
deprivation, Pro-apoptotic Factors
Damage to the mitochondrial membrane increasing permeability
Entry of Cytochrome C into the
cytoplasm
Cytochrome C binds to Apaf-1 forming an
apoptosome
Apoptosome activates procaspase-9 to
caspase-9
Caspase-9 cleaves and activates caspase-3 and
caspase-7.
This executioner caspases activate a cascade of proteolytic activity that leads
to: Chromatin condensation, DNA fragmentation, Protein cleavage,
Membrane permeability
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46. Physiological Intrinsic
receptor pathway damage pathway
MITOCHONDRIAL SIGNALS
Caspase cleavage cascade
Orderly cleavage of proteins and DNA
CROSSLINKING OF CELL CORPSES; ENGULFMENT
(no inflammation)
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47. Steps
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48. Disorders of apoptosis
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49. Apoptosis: Role in Disease
TOO MUCH: Tissue atrophy
TOO LITTLE: Hyperplasia
Neurodegeneration
Thin skin
etc
Cancer
Athersclerosis
etc
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50. Apoptosis: Role in Disease
Neurodegeneration
oNeurons are post-mitotic (cannot replace themselves; neuronal stem cell

replacement is inefficient)
oNeuronal death caused by loss of proper connections, loss of proper growth
factors (e.g. NGF), and/or damage (especially oxidative damage).
oNeuronal dysfunction or damage results in loss of synapses or loss of cell

bodies (synaptosis, can be reversible; apopsosis, irreversible)
oPARKINSON'S DISEASE
oALZHEIMER'S DISEASE
oHUNTINGTON'S DISEASE etc.
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51. Apoptosis: Role in Disease
Cancer
oApoptosis eliminates damaged cells (damage => mutations =>
cancer
oTumor suppressor p53 controls senescence and apoptosis
responses to damage.
oMost cancer cells are defective in apoptotic response(damaged,
mutant cells survive)
oHigh levels of anti-apoptotic proteins
or
oLow levels of pro-apoptotic proteins ===> CANCER
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52. Apoptosis: Role in Disease
Cancer
Virus associated cancer
•Several human papilloma viruses (HPV) have been
implicated in causing cervical cancer. One of them
produces a protein (E6) that binds and inactivates the
apoptosis promoter p53.
•Epstein-Barr Virus (EBV), the cause of mononucleosis
and associated with some lymphomas
–produces a protein similar to Bcl-2
–produces another protein that causes the cell to
increase its own production of Bcl-2. Both these
actions make the cell more resistant to apoptosis
(thus enabling a cancer cell to continue to proliferate).
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53. •Some B-cell leukemia and lymphomas express high
levels of Bcl-2, thus blocking apoptotic signals they may
receive. The high levels result from a translocation of the
BCL-2 gene into an enhancer region for antibody
production.
•Melanoma (the most dangerous type of skin cancer)
cells avoid apoptosis by inhibiting the expression of the
gene encoding Apaf-1.
Apoptosis: Role in Disease
Cancer
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54. •Other cancer cells express high levels of FasL, and can
kill any cytotoxic T cells (CTL) that try to kill them
because CTL also express Fas (but are protected from
their own FasL).
•Some cancer cells, especially lung and colon cancer
cells, secrete elevated levels of a soluble "decoy"
molecule that binds to FasL, plugging it up so it cannot
bind Fas. Thus, cytotoxic T cells (CTL) cannot kill the
cancer cells
Apoptosis: Role in Disease
Cancer
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55. Apoptosis: Role in Disease
Aging
Aging --> both too much and too little apoptosis
(evidence for both)
Too much (accumulated oxidative damage?)
---> tissue degeneration
Too little (defective sensors, signals?
---> dysfunctional cells accumulate
hyperplasia (precancerous lesions)
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56. Apoptosis: Role in Disease
•Apoptosis and AIDS

Hallmark- the decline in the number of the
patient's CD4+ T cells (normally about 1000
per microliter (µl) of blood).
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57. In Immune system
oVery rarely humans are encountered with
genetic defects in apoptosis.
oThe most common one is a mutation in the
gene for Fas
omutations in the gene for FasL or even one of
the caspases are occasionally seen.

Autoimmune lymphoproliferative syndrome
or ALPS.
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58. Conclusion
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59. Cells are balanced between life and death
DAMAGE Physiological death signals
DEATH SIGNAL
PROAPOPTOTIC
PROTEINS
(dozens!)
ANTIAPOPTOTIC
PROTEINS
(dozens!)
DEATH
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60. Thank u….
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