Content- 1. Background 2. Introduction 3. Difference between apoptosis and necrosis 4. Apoptosis in biologic processes 5. Apoptosis in pathologic processes 6. Morphologic features 7. Techniques to identify and count apoptotic cells 8. Biochemical changes 9. Molecular mechanism of apoptosis 10. Recent advancement and emerging trends in apoptosis 11. References

1. Presented by: Ajay Kumar Pal
M.Pharm (Pharmacology)
IInd Semester
Dept. Of Pharmaceutical Sciences
Guided By : Mr. Virendra Tiwari
1
APOPTOSIS – RECENT
ADVANCES

2. 2
 Background
 Introduction
 Difference between apoptosis and necrosis
 Apoptosis in biologic processes
 Apoptosis in pathologic processes
 Morphologic features
 Techniques to identify and count apoptotic cells
Contents

3. 3
 Biochemical changes
 Molecular mechanism of apoptosis
 Recent advancement and emerging trends in apoptosis
 References

4. 4
 Normal cells have a fairly narrow range of function or steady
state: Homeostasis
 Excess physiologic or pathologic stress may force the cell to a
new steady state: Adaptation
 Too much stress exceeds the cell’s adaptive capacity: Injury
Background

5. 5
Background Cont’d...

6. 6
 Cell injury is a sequence of events that occur if the limits of
adaptive capability are exceeded or no adaptive response is
possible.
Background Cont’d...

7. 7
Background Cont’d...

8. Irreversible Injury
8

9. 9
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

10. 10
APOPTOSIS NECROSIS
Role for mitochondria and
cytochrome C
No 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

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APOPTOSIS NECROSIS
Regulatable process Not regulated
Evolutionarily conserved Not conserved
Dead cells ingested by neighboring
cells
Dead cells ingested by neutrophils
and macrophages

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 The process of programmed cell death.
 Biochemical events lead to characteristic cell changes (morphology)
and death. These changes include blebbing, cell shrinkage, nuclear
fragmentation, Chromatin condensation, and Chromosomal DNA
fragmentation.
 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.
APOPTOSIS

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 Definition: A pathway of cell death induced by tightly
regulated intracellular program in which cells destined to die
activate enzymes that degrade cell’s own DNA and nuclear
and cytoplasmic proteins.
APOPTOSIS

14. 14
German scientist Carl Vogt - Principle
of apoptosis (1842)
Walther Flemming – Process of
programmed cell death (1845)
John Foxton Ross Kerr – Distinguished
apoptosis from traumatic cell death (1962)
History

15. 15
Apoptosis in physiologic
situations
Apoptosis in pathologic
situations
APOPTOSIS

16. States Of Apoptosis
16

17. 17
APOPTOSIS IN
PHYSIOLOGICAL
CONDITIONS

18. 18
 Organised cell destruction in sculpting of
tissues during development of embryo.
APOPTOSIS DURING
EMBRYOGENESIS
Formation of
free and
independent
digits
Development
of the brain
Development
of
reproductive
organs

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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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Involution of hormone-
dependent tissues upon
hormone withdrawal

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 Endometrial cell breakdown during menstrual cycle
 Ovarian follicular atresia in menopause.
 Regression of the lactating breast after weaning.
 Prostatic atrophy after castration.

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Cell loss in proliferating
cell populations

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 Immature lymphocytes in bone marrow and thymus that fail
to express useful antigen receptors.
B lymphocytes in germinal centers.
Epithelial cells in intestinal crypts.
So as to maintain a constant number.

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APOPTOSIS IN
PATHOLOGICAL
CONDITIONS

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• Defective apoptosis and
increased cell survivalTOO LITTLE
• Increased apoptosis and excess
cell deathTOO MUCH

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 Cell death in tumours exposed to chemotherapeutic agents.
 Cytotoxic T cell induced cell death such as in immune
rejection and graft versus host disease.
 Progressive depletion of CD4+ T cells in the pathogenesis of
AIDS.
 In degenerative disease of CNS i.e. Alzheimer’s disease,
Parkinson’s disease and chronic infective dementias.

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 Pathologic atrophy of organs and tissues on withdrawal of
stimuli. Ex: Prostatic atrophy after orchiectomy, atrophy of
kidney or salivary gland on obstruction of ureter or ducts
respectively.
 Cell death that occurs in heart diseases such as myocardial
infarction.

28. Morphologic Features
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 Involvement of single cells or small clusters of cells in the
background of viable cells.

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 The apoptotic cells are round to oval shrunken masses of
intensely eosinophilic cytoplasm( mummified cell) containing
shrunken or almost normal organelles.
 The nuclear chromatin is condensed or fragmented (Pyknosis
or Karyorehexis).

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31. 31
 The cell membrane may show convolutions or projections on
the surface.
 Formation of membrane bound near spherical bodies on or
around the cell called Apoptotic bodies containing compacted
organelles.
 Characteristically, unlike necrosis, there is no acute
inflammatory reaction around apoptosis.
 .

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 Phagosytosis of apoptotic bodies by macrophages takes place
at a varying speed.

33. Identification Of Apoptosis
33
 Staining of chromatin condensation.
 Flow cytometry to visualize rapid cell shrinkage.
 DNA changes detected by In-Situ techniques or by gel electrophoresis.
 AnnexinV as marker for apoptotic cell membrane having
phosphatidylserine on the cell exterior.

34. Biochemical Changes
34
 Activation of caspases
 Proteolysis of cytoskeletal proteins.
 Fregmentation of nuclear chromatin by activation of nucleases.
 In some forms of the apoptotic appearance of an adhesive
glycoprotein Thrombospondin on the outer surface of apoptotic
bodies.

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1) Sensors – BAD , BIM , BID,
noxa, puma
2) Proapoptotic- BAX , BAK
3) Cytochrome C
4) Apaf-1 [ apoptosis activating
factor – 1 ]
5) Caspases
1) Antiapoptotic - BCL 2,
BCL XL, MCL-1
2) IAPS: [inhibitors of
apoptosis]

36. CASPASES
36
 A class of cysteine–aspartyl proteases.
 Synthesized as inactive precursor enzymes, or proenzymes.
 Typically lie dormant in the healthy cell.
 In response to cell death stimuli are converted, either by proteolytic
cleavage or by recruitment into large complexes, into active
Caspases are central initiators and executioners of apoptosis.
The fundamental events in apoptosis is the activation of enzymes
called CASPASES.

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enzymes.
 Once activated, caspases cleave their substrates typically after
conserved aspartate residues and are responsible for most of the
biochemical and morphological features of apoptotic cell death.

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There are 14 different caspase enzymes.
Active cysteine residue in the
catalytic site
Synthesized as inactive zymogens
(PROCASPASES)

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 Caspases in their pro-enzyme form contain three domains:
1. An amino-terminal pro-domain
2. A large subunit containing the active site cysteine within a
conserved QACXG motif and,
3. A carboxy-terminal small subunit.
 The prodomains are separated from the large subunit by an aspartate
cleavage site and one or more aspartate cleavage sites are present in
the linker region between the large and small subunits.

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41. 41

42. 3 TYPES OF CASPASES
42
 Inflammatory Caspases: 1, 4, and 5
 Initiator Caspases: 2, 8, 9, and 10
 Effector Caspases: 3, 6, and 7

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Initiator Caspases: 2, 8, 9, and 10
 Long N-terminal domain that allow them to interact with
death effector domains (DED) or caspase recruitment
domains (CARD) present in adaptor proteins such as
FADD(Fas associated protein with death domain) and
Apaf-1respectively.
 Interact with effector caspases.

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Executioner/ Effector Caspases: 3, 6, and 7
 Devoid of death effector domain
 Execute apoptosis/cell death ultimately.
Inflammatory Caspases: 1, 4, and 5
 Characterized by the presence of a CARD domain at the
N-terminus.

45. Function Of Caspases
45

46. Bcl-2 Proteins
46
 Proteins encoded by Bcl-2 gene.
 Bcl-2 which derives its name from B-cell lymphoma 2,
 Oncogenes Bcl-2 found to inhibit cell death, rather than
promote cell proliferation.
 All the Bcl-2 members are located on the outer mitochondrial
membrane.

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 The Bcl-2 family members are divided into three categories
of proteins according to their structure and function:
1. Anti-apoptotic members.
2. Multi-domain pro-apoptotic members.
3. BH3-only pro-apoptotic members.

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49. Mechanism Of Apoptosis
49
 Death Pathway/ Death Receptor pathway/Extrinsic Pathway.
 Mitochondrial Pathway/ Intinsic Pathway.
 Common pathway.
 The intrinsic endoplasmic reticulum pathway/ Endoplasmic
Reticulum Stress induced pathway.

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Death
Ligands
Effector
Caspase 3
Death
Receptors
Initiator
Caspase 8
Cell
death
DNA
damage
& p53
Mitochondri
a/Cytochrom
e C
Initiator
Caspase 9
“Extrinsic Pathway”
“Intrinsic Pathway”
Apoptotic Pathway (General Outline)

51. Extrinsic Pathway
51
 The extrinsic pathway occurs when there is binding of a so-
called ‘‘death ligand’’ to a cell surface receptor, such as
Fas/CD95/Apo-1, a member of the tumor necrosis factor
(TNF) family of apoptosis-inducing receptors.
 Binding of trimeric Fas ligand to the Fas receptor triggers
oligomerization and formation of a death-inducing signaling
complex (DISC) comprised of Fas, the adaptor molecule
FADD and procaspase-8.

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 Formation of DISC induces conformational changes in
procaspase and cleavage of pro-caspase from its pro-domain
leads to activation of caspases.
 Caspase 8 is an initiator caspase, which initiates apoptosis by
cleaving other downstream or executioner pro-caspases.
 Caspase 8 activation intiates caspase cascade activation and
finally caspase-3 activation causes cell death, by inducing
apoptotic morphological and biochemical changes.

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 Ultimately, Recognition of apoptotic cell by neighbouring cell
which then ultimately phagocytize it.

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55. Intrinsic Pathway
55
 Initiated within the cell.
 Internal stimuli such as irreparable genetic damage, hypoxia,
extremely high concentrations of cytosolic Ca2+ and severe
oxidative stress are some triggers of the initiation of the
intrinsic mitochondrial pathway.

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Internal stimuli such as irreparable genetic damage, hypoxia, extremely
high concentrations of cytosolic Ca2+ and severe oxidative stress
induces DNA damage as well as mitochondrial membrane.
Triggers initiation of intrinsic
pathway.
Increased mitochondrial permeability and the release of pro-apoptotic
molecules such as cytochrome-c into the cytoplasm from the inter-
membrane space of mitochondria.
Expression of P53 gene and
Bcl-2 family proteins due to
DNA damage.Bax

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Oligomer
of BAX
Intermembr
ane
Mitochondri
al Space
Cytoplasm

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Pro-Caspase 9 then activate and active Caspase 9 form complex with
Apaf-1 portion of Apaptosome.
Cytochrome C bind with Apaf-1 and form Apaptosome.
Cyt c

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Binding of caspase-9 to Apaf-1 Of apatosome induces activation of
Caspase-3

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Caspase-3 ultimately cause cell death.
By inducing cleavage of protein kinases, cytoskeletal proteins,
DNA repair proteins and inhibitory subunits of endonucleases
family.
Effect on the cytoskeleton, cell cycle and signaling
pathways, which together contribute to the typical
morphological changes in apoptosis.

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62. Recent Advances
62
 Salvesen laboratory suggest that only XIAP is a true direct
inhibitor of caspases, the greatest potency for caspase
inhibition compared to the other IAPs.
 XIAPs have been shown to antagonize the apoptotic cascade
via the direct inhibition of caspases and via proteasome-
dependant degradation of caspases.

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Caspase inhibitors:
VX-765 is an orally active, reversible caspase-1 inhibitor
that was being developed for the treatment of inflammatory
disorders.
Emricasan is a novel, irreversible, orally active pan-caspase
inhibitor that has been investigated for the treatment of
chronic HCV infection and liver transplantation rejection.

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 BCL2 inhibitors:
ABT263 is a small molecule mimetic of the BH3 domain of
the pro-apoptotic BAD protein that is currently in clinical
trial in chronic lymphatic leukaemia.
 G3139 is an antisense oligo-deoxynucleotide
targeting BCL2 mRNA resulting in RNAse H activation.
 ABT-737 is highly effective against cancers with elevated

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expression of Bcl-2 and is an efficient apoptosis inducer in the
presence of Bax and Bak in small cell lung cancer mouse model.
NCX-1000, a small-molecule inhibitor that selectively inhibits
caspase-3, 8 and 9 in the micro-molar range, was in phase-II
clinical trials for the treatment of chronic liver disease.
PAC-1, a small molecule that induces both procaspase-3
activation in vitro and apoptosis in several cancer cell lines.

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67. References
67
 Apoptosis: A Review of Programmed Cell Death ---
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2117903/
accessed on 12 March at 9:23 pm.
 C. Soldani and A. I. Scovassi “Poly(ADP-ribose) polymerase-
1 cleavage during apoptosis: An update” Kluwer Academic
Publishers, Apoptosis 2002; 7: 321–328
 Allison M, Eric C, et all, ”The inhibitors of apoptosis (IAPs)
as cancer targets”

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 S. Oyadomari, E. Araki, et all, “Endoplasmic reticulum stress-
mediated apoptosis in pancreatic β-cells” Kluwer Academic
Publishers, Apoptosis 2002; 7: 335–345
 Reed JC: Bcl-2 family proteins: regulators of apoptosis and
chemoresistance in haematologic malignancies. Semin Haematol
1997, 34:9-19.
 Jes´us Gil and Mariano Esteban, “Induction of apoptosis by the
dsRNA-dependent protein kinase (PKR): Mechanism of action”
Kluwer Academic Publishers, Apoptosis 2000; 5: 107–114

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