Apoptosis is a -pathway of cell death that is -induced by an internally regulated program -in which cells destined to die activate intrinsic enzymes that --degrade the cells’ own nuclear DNA and also nuclear and cytoplasmic proteins -With minimal host reaction.

1. Apoptosis-101
Presented on-
Date- 21-11-14
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2. Cell response to stressful conditions
and injurious stimuli
• When limits of adaptive responses are
exceeded or if cells are exposed to
injurious agents or stress, deprived of
essential nutrients, or become
compromised by mutations that affect
essential cellular constituents, a
sequence of events follows that is
termed cell injury
• Cell death, the end result of
progressive cell injury
• two principal pathways of cell death,
– necrosis
– apoptosis
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3. Apoptosis (Greek origin)=“falling off”
• 1842- Vogt gave first principles of apoptosis
• 1885- Flemming gave more precise description
• 1965- John Kerr using EM distinguished it from
traumatic cell death
• 1972- coining of term “apoptosis” by Kerr, Wyllie,
Currie
• 1990 onwards- use of newer methods to study
proteins and genetics has given newer insights
into apoptosis.
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4. Definition
• Apoptosis is a
– pathway of cell death that is
– induced by an internally regulated program
– in which cells destined to die activate intrinsic
enzymes that degrade the cells’ own nuclear DNA
and also nuclear and cytoplasmic proteins
– With minimal host reaction.
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5. Causes
Physiological Pathological
Death by apoptosis is a
normal phenomenon that
serves to eliminate cells
that are no longer needed,
and to maintain a steady
number of various cell
populations in tissues.
Apoptosis eliminates cells
that are injured beyond
repair
without eliciting a host
reaction, thus limiting
collateral
tissue damage.
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6. Apoptosis in physiological conditions
Destruction of cells
during
embryogenesis
Implantation
Organogenesis
Involution of
hormone-dependent
tissues upon
hormone withdrawal
Endometrial cell breakdown
ovarian follicular atresia in menopause,
Cell loss in
proliferating cell
populations
Immature lymphocytes in the bone marrow and thymus and B
lymphocytes in germinal centers that fail to express
useful antigen receptors
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7. Apoptosis in pathological conditions
DNA damage Radiation, cytotoxic anticancer drugs, and
hypoxia can damage DNA. If repair mechanisms cannot
cope with the injury, the cell triggers intrinsic mechanisms
that induce apoptosis. In these situations elimination
of the cell may be a better alternative than risking
mutations in the damaged DNA, which may result in
malignant transformation.
Accumulation of
misfolded proteins
ER stress, which culminates in apoptotic cell death. Apoptosis
caused by the accumulation of misfolded proteins has been
invoked as the basis of several degenerative diseases of the
central nervous system and other organs.
Cell death in
certain infections,
particularly viral
infections
loss of infected cells is largely due to apoptosis that may be
induced by the virus or by the host immune response (as in viral
hepatitis).
Pathologic atrophy
in parenchymal
organs after duct
obstruction
Pancreas, parotid gland, and
Kidney
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8. Morphological changes
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9. Morphological changes characterizing
apoptosis
Cell shrinkage
Chromatin
condensation
Formation of
cytoplasmic
blebs and
apoptotic
bodies
Phagocytosis
of apoptotic
cells
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10. Cell shrinkage
• Cell becomes
smaller in size
• Cytoplasm is dense
• Organelles tightly
packed
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11. Chromatin condensation
• Chromatin aggregates
peripherally under
nuclear membrane in
form of irregular dense
masses (pyknosis)
• The nucleus may break
up into fragments
(karyorrhexis)
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12. Cytoplasmic blebs and apoptotic bodies
• Apoptotic cells
first undergoes
surface blebbing
• Then it fragments
into many
membrane bound
apoptotic bodies
(karyolysis)
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13. Phagocytosis of apoptotic cells
• Phagocytes ingest apoptotic cells and
degradation occurs by lysosomal enzymes.
• Phagocytic cells- macrophages
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14. • A) Low power --cell shrinkage in apoptotic cell
• B) High power --apoptotic cell nucleus showing
chromatin condensation
• In both surrounding cells are normal
A
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16. Mechanism of apoptosis
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17. Apoptosis mechanism
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18. Apoptosis mechanism
• Apoptosis results from the activation of
enzymes called caspases (which exist as
inactive proenzymes, or zymogens).
• Phases of apoptosis-
– Initiation- phase of activation of caspases
– Execution- activated caspases trigger cell
degradation.
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19. Two Pathways of initiation of apoptosis
1. Intrinsic 2. Extrinsic
Mitochondrial Death-receptor
initiated
Initiator phase
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20. • An overview of
the extrinsic
and intrinsic
pathways. Both
pathways lead
to activation of
caspase-3 and
give rise to
apoptotic cell
death.
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21. Intrinsic pathway
• Major mechanism of apoptosis in all mammalian
cells.
• Increased permeability of the mitochondrial
outer membrane with consequent release of
death-inducing (pro-apoptotic) molecules from
the mitochondrial intermembrane space into the
cytoplasm.
• The release of mitochondrial pro-apoptotic
proteins is tightly controlled by the
– BCL2 family of proteins
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22. BCL2 family of proteins
• BCL2 stands for B-cell lymphoma 2.
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23. BCL2 family of proteins
Group Members Details
Anti-apoptotic •BCL2,
•BCL-XL
•MCL1
Present in outer mitochondrial
membrane
Prevent leakage of cytochrome-c and
other death inducing proteins into
cytosol.
Pro-apoptotic •BAX
•BAK
On activation they increase
mitochondrial membrane permeability.
Sensors •BAD
•BIM,
•BID
•Puma, Noxa
Act as sensors of cellular stress and
damage, and regulate the balance
between the other two groups.
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24. • When
– cells are deprived of survival
signals
– DNA is damaged
– misfolded proteins
• Sensor proteins activate
and further activate pro-
apoptotic proteins.
• Also anti-apoptotic factor
production decreases.
• With this several
mitochondrial proteins
(mainly cyt-c) is released
into cytosol.
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25. Cyt-c in cytosol
• Cytochrome c binds to a protein called APAF-1
(apoptosis-activating factor-1), which with
effect of ATP forms the apoptosome.
• Apoptosome activates caspase activation
cascade and leads to execution phase of
apoptosis.
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26. SMAC proteins in cytosol
• Mitochondrial proteins known as SMACs
(small mitochondria-derived activator
of caspases) are released into
the cytosol following an increase in
permeability. SMAC’s neutralize inhibitor of
apoptosis proteins (IAPs).
• This leads to caspase activation thus apoptosis
proceeds to execution phase.
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27. Extrinsic pathways
• This pathway is initiated by engagement of plasma
membrane death receptors on a variety of cells.
• These death receptors belong to TNF receptor family.
• These receptors link with a external protein to form a
complex termed “death domain” which is important
to activate apoptosis.
• Important death receptor activating proteins are
– TNF
– Fas ligand
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28. TNF pathway
• TNF is
– Produced by
macrophages
– Main external signal
of apoptosis
• TNF links with
TNFR1 to form
death domain
termed TRADD
• TRADD activates
caspase 8 which
initiates apoptosis.
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29. Fas pathway
• Fas ligand(FasL) is present
on
– NK cells
– Other T cells
• 3Fas and FasL bind to form
Fas-associated death
domain (FADD)
• This activates pro-caspase
8 to an active form.
• Caspase 8 initiates the
further execution phase.
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30. Execution phase
• Both initiating pathways converge at level of
caspase activation.
• Initiator caspases(8,9,10) activate the
executioner caspases(3,6).
• These executioner caspases signal DNA
cleavage.
• DNA clevage causes disintegration of
nucleus(karyorrhexis) and also damages
nuclear membrane.
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31. Clearing of dead cells
• Clearing of apoptotic cells is the final step of
apoptosis and in healthy individuals this process is
efficient and rapid enough to prevent any
inflammation and necrosis.
• Phagocytic cells- macrophages.
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32. Signals= substances expressed on cell surface
• Finding of apoptotic cells- “find me” signals
– fractalkine, LPC, S1P
• Start of eating of apoptotic cells-”eat me” signals
– phosphatidylserine flip out
– Expression of C1q, thrombospondin
• Viable cells giving out-”don’t eat me” signals
– CD47 expression
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34. • Standard H&E-stained section of lymph node showing
macrophages (arrows) of a germinal centre containing
engulfed remnants of apoptotic cells.aj

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36. • Few examples of apoptosis-
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37. • In this fetal thymus there is involution of thymic lymphocytes by
the mechanism of apoptosis.
• Individual cells fragment and are consumed by phagocytes to give
the appearance of clear spaces filled with cellular debris.aj

38. • Microphotograph shows a later stage of apoptosis(A) in
epithelial cells of endometrial glands at the beginning of
menstruation. aj

39. • Cell turnover in crypts of colonic epithelium
occurs by apoptosis.
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40. • Corpus luteum, formed from an ovarian follicle after discharge of an ovum.
Unfertilised ovum, the corpus luteum will involute, a process that involves
progressive death of its constituent cells, leaving a fibrotic scar known as a
corpus albicans.
• In this micrograph several apoptotic cells AC can be identified by their
condensed nuclei and eosinophilic cytoplasmaj

41. • Apoptosis of two neutrophils in normal anticoagulated blood
during standing at room temperature. Nuclear condensation
and fragmentation are evident. A normal neutrophil is also
present.
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43. • Apoptosis of an epidermal cell in an immune reaction(GVHD).
• The cell is reduced in size and contains brightly eosinophilic
cytoplasm and a condensed nucleusaj

44. • Councilman hyaline body, is an eosinophilic globule often
surrounded by normal parenchyma found in the liver of individuals
suffering from viral hepatitis, yellow fever, or other viral syndrome.
• It represents a hepatocyte that is undergoing apoptosis.aj

45. • Erythema Multiforme (EM) is a hypersensitivity reaction
usually caused by infections, mostly Herpes simplex virus.
• Keratinocytes show apoptosis.
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46. Dysregulated apoptosis
-too less
-too much
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47. Defective (less than normal)apoptosis in cancer
• Cancer cells resist apoptosis by
– Gaining mutated TP53
– Producing more anti-apoptotic factors
• TP53
– In normal conditions causes DNA repair or if
damage is irrepairable it causes apoptosis.
– In most human cancers the causative oncogenic
viruses inactivate TP53 by binding to it.
– Due to this excess cell proliferation forms a tumor
mass.
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48. Excessive apoptosis
• Neurodegenerative diseases
– Apoptosis of specific set of neurons having
mutations or misfolded proteins.
– Eg. Alzheimer’s disease, CJ disease, cystic fibrosis
• Death of viral infected cells in many viral
infections.
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49. Apoptosis vs necrosis
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51. New concepts-
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52. Assays for apoptosis
• Aim- to find the fragmented DNA
• Demonstration of nucleosomal “laddering”
which is diagnostic of DNA fragmentation
– This method uses electrophoresis
• TUNEL assay
– This method uses flow cytometry
• Gold standard for detecting apoptosis in fixed
tissue is electron microscopy
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54. Thank you
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