apoptosis and recent advances,intrinsic and extrinsic pathway pyroptosis, necroptosis

1. APOPTOSIS –
RECENT ADVANCES
EKTA JAJODIA

2. •German scientist Carl Vogt -
Principle of apoptosis (1842)
•Walther Flemming – Process of
programmed cell death (1845)

3. John Foxton Ross Kerr –
Distinguished apoptosis from
traumatic cell death (1962)

4. INTRODUCTION

5. Apoptosis is an energy dependent programmed cell
death for removal of unwanted cells
DEFINITION

6. Cell death mechanisms
Death by suicide Death by injury
sclero dinesh

7. 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

8. 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
Regulatable process Not regulated
Evolutionarily conserved Not conserved
Dead cells ingested by
neighboring cells
Dead cells ingested by
neutrophils and macrophages

9. Apoptosis in physiologic situations
Apoptosis in pathologic situations
APOPTOSIS

10. APOPTOSIS IN
PHYSIOLOGICAL
CONDITIONS

11. Formation of
free and
independent
digits
Development
of the brain
Development
of
reproductive
organs
APOPTOSIS
DURING
EMBRYOGENESIS

12. 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

13. Involution of
hormone-dependent
tissues upon
hormone withdrawal
•Endometrial cell breakdown during menstrual cycle
•Ovarian follicular atresia in menopause
•Regression of the lactating breast after weaning
•Prostatic atrophy after castration

14. Cell loss in
proliferating cell
populations
•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

15. Elimination of
potentially harmful
self-reactive
lymphocytes

16. Death of host cells
that have served
their useful purpose
•Neutrophils in an a/c inflammatory response
•Lymphocytes at the end of an immune response

17. APOPTOSIS IN
PATHOLOGICAL
CONDITIONS

18. DNA
damage
Accumulation
of mis-folded
proteins
Cell injury in
certain
infections
Pathological
atrophy in
parenchymal
organs after
duct obstruction

19. • Defective apoptosis
and increased cell
survival
TOO
LITTLE
• Increased apoptosis
and excess cell
death
TOO
MUCH

20. Unfolded protein
response(UPR) and
Endoplasmic
reticulum(ER) stress

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

24. The fundamental events in apoptosis is the activation of
enzymes called CASPASES
• Cysteine proteases
• Cysteine-dependent
ASPartate-specific
proteASES
Caspases
CASPASES

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

27. • Inflammatory Caspases: 1, 4, 5 and 13
• Initiator Caspases: 2, 8, 9, and 10
– Long N-terminal domain
– Interact with effector caspases
• Effector Caspases: 3, 6, 7 and 14
– Little to no N-terminal domain
– Initiate cell death
3 TYPES OF
CASPASES

28. Procasp 2 & 9
have CARD
domain
Procasp 8 &
10 have DED
domain
In intrinsic
pathway
Procasp 9
Procasp 9
activated by
apoptosome
in extrinsic
pathway
Procasp 8 &
10
Procasp 8 &
10 activated
by death
receptors
2
8
9
10
CARD – caspase recruitment domain
DED – death effector domain

29. 3
6
7
procaspase 3 and 7
activated by granzyme B
14

30. BCL-2
PROTEINS
Proapoptotic
proteins
• BH123 protein
• BAX
• BAK
Antiapoptotic
proteins
Bcl2
BclXL
Mcl-1
Sensors/proapop
totic proteins
• Bad
• Bid
• Bim
• Puma
• Noxa

35. DAMAGE Physiological death signals
DEATH SIGNAL
PROAPOPTOTIC
PROTEINS
ANTIAPOPTOTIC
PROTEINS

36. INHIBITORS OF
APOPTOTIC PROTEINS
(IAPs)
•1st identified in baculovirus
•8 types are described
•IAPs are characterised by BIR domains (baculoviral IAP
repeats)
•These BIR domains bind to caspases and inhibit them
•IAPs are regulated by IAP binding proteins – Smac/DIABLO
(second mitochondrial activator of caspases)

37. NAIP – neuronal apoptosis inhibitor protein
Cellular IAP1
Cellular IAP2
XIAP – X-linked inhibitor of apoptosis
Survivin
Ts-IAP – testis specific IAP
BRUCE – BIR containing ubiquitin conjugating enzyme
Livin

38. Normally Smac/DIABLO resides in mitochondria(intermitochondrial
space)
When apoptotic stimulus triggers
Smac/DIABLO is proteolytically cleaved and released into cytosol
Bind to IAPs and inhibit them
Causes apoptosis

39. XIAP has been best described
Inhibits apoptosis in 2 ways
Direct inhibition of
apoptosis by binding
of BIR domains to
caspases
Proteosome dependent
degradation of caspases

40. • Mitochondrial protein present at inter mitochondrial
space
• Can activate caspase cascade
• Binds to APAF-1 and form apoptosome
CYTOCHROME C

41. [ apoptosis activating factor – 1 ]
• Present in cytosol
• Forms apoptosome after combining with
cytochrome c
APAF-1

42. Initiation
• Intrinsic pathway
• Extrinsic Pathway
Execution
• Cleavage of DNA
Phagocyt--
osis
• Engulfment of cell
PATHWAYS OF
APOPTOSIS

43. INTRINSIC PATHWAY
(MITOCHONDRIAL)

44. INTRINSIC PATHWAY [MITOCHONDRIAL PATHWAY]
Radiation , Toxins, Free radicals
misfolded proteins/ DNA damage
ER STRESS Sensing of double strand DNA break

45. Double strand DNA break is sensed by 2
proteins
ATM (Ataxia telangiectasia
mutated)
ATR (ataxia telangiectasia and
RAD3 related protein
Phosphorylates and activates CHK1 and CHK2
(checkpoint kinase 1 and 2)
Phosphorylates and activates P53

46. P53 form tetramers with other p53 molecules and
cause increase expression of the following genes
Genes involved in DNA repair
P21 genes
Proapoptotic proteins/sensors
(puma/noxa)

49. Activation of proapoptotic proteins (BAX
and BAK)
Oligomerisation and increase in outer
mitochondrial membrane permeability
Leakage of cytochrome c
into cytosol
Release of Smac/DIABLO into
cytosol which binds to IAPs and
inhibits them

50. Cytochrome c in cytosol + APAF 1
Form Hexamer ( Apoptosome )
Binds to procaspase- 9
( critical initiator caspase)
Enzyme cleaves the adjacent caspase 9 molecules
i.e . Autoamplification process
Execution phase

51. EXTRINSIC
PATHWAY

53. Death Receptors
Members of TNF receptor family which contain a
cytoplasmic domain involved in protein-protein
interactions .i.e called Death Domain .
2 types of death receptors are there :
• TNFr (tumour necrosis factor receptor )
• FasR (fatty acid synthetase receptor )
The ligand for Fas is called FasL
The ligand for TNFr is TNFα
Adaptor Proteins
also contain death domain
a. FADD [ Fas associated death domain ]
b. TRADD [ TNF receptor associated death domain]

54. The death
receptor
pathway
• Extrinsic
pathway
Inhibited by FLIP- binds to
procaspase8 but cannot cleave
and activate it

56. EXECUTION PHASE
• Final phase of apoptosis
• Mediated by proteolytic cascade
• After initiator caspases ( 2, 8, 9 and 10) are cleaved to
generate its active form, the enzymatic death program
is set in motion by rapid sequential activation of the
executioner caspases – 3,6
• Caspase -3 activates DNase which causes degradation
of chromosomal DNA within the nuclei and causes
chromatin condensation.
• Caspase -3 induces cytoskeletal reorganisation and
disintegration of cell into apoptotic bodies.

58. REMOVAL OF DEAD CELLS
Factors by which apoptotic cells attracts
phagocytes towards them :
• Apoptotic bodies- “bite size”-edible for
phagocytes
• Phosphatidyl serine “flips” out from inner to
outer layer –recognized by macrophages
receptors
• Some apoptotic bodies express thrombospondin
recognized by phagocytes

59. APOPTOSIS
ASSAYS- detection
and methods

60. Cell Viability and Death
• Functional assay
• DNA labeling assay
• Morphological assay
• Reproductive assay
• Membrane integrity assay

61. • Exclusion dyes
• Flourescent dyes
• annexinV
• LDH assay
MEMBRANE
INTEGRITY ASSAY
• MTT, XTT assay
• Crystal violet/ Acid
• phosphatase(AP) assay
• Alamar Blue oxidation-Reduction
assay
• Neutral red assay
FUNCTIONAL
ASSAY
• Fluorescent conjugates
DNA LABELLING
ASSAY

62. • Microscopic observation
• -Caspase 3 detection
• -PARP cleavage assay
MORPHOLOGICAL
and MECHANISM
BASED ASSAY
• Colony formation assay
REPRODUCTIVE
ASSAY
• annexinV
• Caspase3
• p53
IMMUNOHISTOCHEMIS
TRY

63. MEMBRANE
INTEGRITY ASSAY
•Exclusion dyes
•Flourescent dyes
•AnnexinV
•LDH assay

64. EXCLUSION
DYES
• Viable cells : small, round and refractile
• Non-viable cells : swollen, larger, dark blue
• Tryphan blue dye exclusion methods

65. Principle
Trypan Blue dye Exclusion Methods

66. • Ethidium bromide (EtBr) and propidium iodide (PI)
• PI binds to nucleic acids upon membrane damage
• PI is impermeable to intact plasma membrane.
• Intercalates with DNA or RNA  red
FLUORESCENT
DYES

67. Fluorescein diacetate (FDA) is a nonpolar ester which
passes through plasma membranes and is hydrolyzed by
intracellular esterases to produce free fluorescein
 the polar fluorescein is confined within cells which have
an intact plasma membrane and can be observed under
appropriate excitation conditions
Undamaged cell : highly fluorescent fluorescein dye
Damaged cell : fluoresce only weakly
 greenish-yellow at 450-480 nm

68. Intact cell –
PI and FDA is added
Fluorescein in
intact cells
Schematic illustration of the principle of
PI/FDA cell viability assay
● FDA (Fluorescein diacetate)
● PI (Propidium iodide)
Plasma membrane is damaged
; fluorescein leaks out
PI enters and stains
nucleic acids

69. ANNEXIN V
ASSAY

70. • Annexin V: An Early Marker of Apoptosis
• One of the earliest indications of apoptosis is the
translocation of the membrane phospholipid
phosphatidylserine (PS) from the inner to the outer
leaflet of the plasma membrane.
• Once exposed to the extracellular environment, binding
sites on PS become available for Annexin V, a 35-36 kDa,
Ca 2+-dependent, phospholipid binding protein with a
high affinity for PS.
• The translocation of PS precedes other apoptotic
processes such as loss of plasma membrane integrity,
DNA fragmentation, and chromatin condensation.

71. • As such, Annexin V can be conjugated to biotin or to a
fluorochrome such as FITC, PE, APC, Cy5, or Cy5.5, and
used for the easy, flow cytometric identification of cells
in the early stages of apoptosis
• Because PS translocation also occurs during necrosis,
Annexin V is not an absolute marker of apoptosis.

72. • Therefore, it is often used in conjunction with vital dyes
such as 7-amino-actinomysin (7-AAD) or propidium
iodide (PI), which bind to nucleic acids, but can only
penetrate the plasma membrane when membrane
integrity is breached, as occurs in the later stages of
apoptosis or in necrosis
• Result
• annexin-/PI-, annexin +/PI-, annexin+/PI+

73. No Apoptosis = Cell Viability
Cells that are negative for both Annexin V and the vital
dye have no indications of apoptosis: PS translocation has
not occurred and the plasma membrane is still intact.
Early Apoptosis
Cells that are Annexin V-positive and vital dye-negative,
however, are in early apoptosis as PS translocation has
occurred, yet the plasma membrane is still intact.

74. Late Apoptosis or Cell Death
Cells that are positive for both Annexin V and the vital dye
are either in the late stages of apoptosis or are already
dead, as PS translocation has occurred and the loss of
plasma membrane integrity is observed.
When measured over time, Annexin V and a vital dye can
be used to monitor the progression of apoptosis: from cell
viability, to early-stage apoptosis, and finally to late-stage
apoptosis and cell death.

77. LDH LEAKAGE
ASSAY
Test principle
The assay is based on consideration that tumor cells
possess high concentration of intracellular LDH and the
cleavage of a tetrazolium salt when LDH is present in the
culture supernatant.

78. • LDH catalyzes the reduction of NAD+ to NADH and H+ by
oxidation of lactate to pyruvate. In the second step of
the reaction, diaphorase uses the newly-formed NADH
and H+ to catalyze the reduction of a tetrazolium salt
(INT) to highly-colored formazan which absorbs strongly
at 490-520 nm.

80. FUNCTIONAL
ASSAY
•MTT, XTT assay
•Crystal violet/ Acid
•phosphatase(AP) assay
•Alamar Blue oxidation- Reduction assay
• Neutral red assay

81. MTT ASSAY
Introduction
 This assay is a sensitive, quantitative and reliable
colorimetric assay that measures viability, proliferation
and activation of cells.
 The assay is based on the capacity of mitochondrial
dehydrogenase enzymes in living cells to convert the
yellow water-soluble substrate dimethylthiazol
tetrazolium bromide (MTT) into a dark blue formazan
product which is insoluble in water

82.  The amount of formazan produced is directly proportional
to number of viable cells present in the sample.
metabolically active Cell
MTT
Formazan
Insoluble

83. Compare with MTT assay and XTT assay
Culture cells in a MTP
for a certain period of time (37℃)
MTT assay XTT assay
Prepare labeling mixture
Incubate cells (0.5-4 h, 37℃)
Add solubilizing solution
(Isopropanol) and incubate
Measure absorbance using an ELISA reader
Add XTT labeling mixtureAdd MTT labeling reagent
Insoluble formazan Soluble formazan

84. DNA LABELLING ASSAY
•Fluorescent conjugates

85. TUNEL ASSAY

87. •The single cell gel electrophoresis (SCGE), the Comet
Assay, is a fairly simple procedure by using a micro gel
and electrophoresis to detect DNA damage
•The damage in DNA looks like a comet, so that’s why it is
known as Comet Assay
• This technique is further developed and introduced the
use of high alkaline conditions. This step increased the
ability of the assay to detect not only the double strand
breaks but also the single strand breaks.
COMET ASSAY

89. MORPHOLOGICAL
MECHANISM BASED
ASSAY
•Microscopic observation
•-Caspase 3 detection
•-PARP cleavage assay
•Endonuclease assay

90. Principle
During apoptosis the endonuclease enzymes are activated
to cleave the genomic DNA in to many smaller fragments
ENDONUCLEASE
ASSAY

91. Steps
1. Preparation of agarose gel containing
Ethidium bromide and genomic DNA
2. Making well
3. Preparation of cell lysate
4. Incubation in humidified atmosphere
after loading cell lysate
5. Observe the DNA degradation

92. MORPHOLOGY OF CELLS IN APOPTOSIS
• Cell Shrinkage and dense cytoplasm
• Chromatin condensation to periphery and later
fragmentation.
• Plasma membrane is intact
• Formation of cytoplasmic blebs and apoptotic
bodies.
• Structurally altered so that the apoptotic cell
becomes “tasty” for phagocytosis
• Phagocytosis of apoptotic bodies by macrophages
• Dead cell is rapidly cleared before contents are
leaked out
• No inflammatory reaction

93. MORPHOLOGY OF APOPTOSIS
Progressive cell shrinkage
Chromatin condensation
Plasma membrane blebbing
Apoptotic bodies
Phagocytosis - no inflammation

94. MORPHOLOGY OF CELLS IN APOPTOSIS

95. Various methods available for
detection of apoptotic cells
Light microscopy
• In H&E the apoptotic cell appears as round or
oval mass of intensely eosinophilic cytoplasm
with fragments of dense nuclear chromatin

97. MICROSCOPY
Apoptosis in neutrophils

98. Councilman bodies- liver tissue

99. Electron microscopy
Defines subcellular changes
like
• Chromatin condensation
• Plasma membrane
blebbing

100. Immunohistochemistry
Immunohistochemical
detection of apoptotic cells
using antibodies against a
wide range of substrates
most importantly :
• Caspase 3
• P53
• Annexin V

101. NECROPTOSIS
•Mechanistically resembles apoptosis
•Morphologically resembles necrosis
•Also known as programmed necrosis
•CASPASES ARE NOT ACTIVATED
•RIp1 and RIP3 complex formed
•Inflammation present

103. PYROPTOSIS

104. Thank u….