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Apoptosis and necrosis

Cell death
Cells die by one of two mechanisms necrosis or
apoptosis
Two physiologically different processes
Necrosis death by injury
Apoptosis death by suicide
Apoptosis and necrosis have different characteristics
Necrosis
Death by accident
Associated with nonphysiological circumstances that
disrupt cellular homeostasis (eg., ischemia, hypoxia and
poisoning)
Necrosis is caused by membrane dissolution (osmotic lysis,
shear stress, pore-forming proteins, loss of ATP)
Necrosis is bad because cellular material (including
degradative enzymes) is released into surrounding tissue
Affects contiguous groups of cell
Necrosis usually causes inflammatory reaction
Cytological characteristics of necrosis
Initial swelling of the cell
Rupture of the plasma
membrane
Cytoplasm is spilled to the
extracellular environment
Types of Necrosis
Coagulation Necrosis
Seen in infarcted organs, e.g.myocardial infarct
Liquefaction Necrosis
Softening of the center of an abscess
Caseous Necrosis
Cheesy, crumbly appearance, e.g. tuberculosis lesion in
the lung
Apoptosis
Death by design genetically programmed cell death
Induced by new gene synthesis, primarily in response to
developmental cues
Requires new RNA and protein synthesis
Inhibitors of transcription or translation prevent
apoptosis
Important for development, homeostasis and elimination
of pathogens and tumor cells
Causes deletion of individual cells in the midst of others
But it can be involved in deletion of entire structures
Apoptosis is followed by fast phagocytosis
Anti-inflammatory (housekeeping)
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Apoptosis
This is not death with the headlights on and the traffic
stopped; rather it is akin to the night-time anatomical
grave-robbing of old, except that for apoptosis the body
may be dismembered, but is not quite dead
Morphologic changes during apoptosis
Membranes become irregular
Chromatin becomes condensed and
segregated
Condensation of cytoplasm
DNA is fragmented
Cell is fragmented and phagocytosed
Apoptotic cell
caspases
induction
execution
degradation
point of no return
Death receptors
Growth factor deprivation
ceramide
Genotoxic insults
Chemotherapeutic drugs
casp9
m
Bcl2
H
2
O
2
UV
Apaf1
AT
P
Membrane blebbing
Cell shrinkage
Chromatin condensation
DNA fragmentation
The three phases of apoptosis
Mechanism of apoptosis
Internal signals mitochondrial pathway
External signals death receptor pathway
Apoptosis inducing factor
Why die by apoptosis?
apoptosis necrosis
Spillage of cell content
Provoke cytokine secretion
Inflammatory response
No spillage of cell content
Apoptotic bodies engulfed by phagocytes
No inflammatory response
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Features of apoptosis vs. necrosis
Chromatin condensation
Cell shrinkage
Preservation of organelles
and cell membranes
Rapid engulfment by
neighboring cells
preventing inflammation
Biochemical hallmark -
DNA fragmentation
Nuclear swelling
Cell swelling
Disruption of organelles
Rupture of cell and release
of cellular contents
Inflammatory response
Apoptosis Necrosis
membrane
blebbing &
changes
mitochondrial
leakage
organelle
reduction
cell
shrinkage
nuclear
fragmentation
chromatin
condensation
APOPTOSIS: morphology
Morphological features of apoptosis
Membrane blebbing
Bleb
Blebbing & Apoptotic bodies
The control retained over the cell
membrane & cytoskeleton allows intact
pieces of the cell to separate for
recognition & phagocytosis
Apoptotic body
DNA fragmentation and gel
electrophoresis
Digestion of DNA starts after 2 hrs
3&4 hrs after initiation of apoptosis
DNA is almost all degraded
DNA is fragmented with restriction
endonucleases
Apoptosis induces 180 bp laddering
of DNA
Targets of endonuclease attack -
linker regions between nucleosomes
Nuclear
breakdown
(Hoechst)
Other morphological features of
apoptosis
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Apoptotic body
M
M
membrane changes
Call in the macrophages with a surface marker
Plasma membrane changes:
Phosphatidylserine is exposed externall y
Membrane proteins lose their normall y
asymmetric distribution across the membrane
Macrophage recognition of the
apoptotic cell
Phosphatidylserine
externalisation
phosphatidylethanolamine phosphatidylserine phosphatidylcholine sphingomyelin
Detection
Annexin V binding
Engulfment signals
Necrosis
The disorganization is primarily
of the plasma & organelle
membranes because of a power
failure - the energy supply for
the ion pumps
Net result - cell swelling &
lysis/dissolution
Mitochondria
Lysosomal spillage
Cell swelling
Loss of cristae
Swelling
Disintegration
Ribosomes dispersed
ER dilated & dissol ved
Chromatin unevenl y clumped
Cytoskeleton eaten up
Necrosis
membranes intact
invites phagocytosis
shrinkage
remains controlled
NORMAL
membranes leaky
spillage
whole cell dissolved
largely nuclear
inflammatory
Apoptosis vs. necrosis Techniques to recognize apoptosis
Morphological assessment
Measurement of tissue transglutaminase
Measurement of DNA fragmentation
Flow cytometry of apoptotic cells
Specific probes for apoptotic cells Annexin V
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control apoptotic
Morphological assessment
Light microscopy
DNA condensation, nuclear
fragmentation, apoptotic bodies
Electron microscopy
Scanning EM
Transmission EM
Tissue transglutaminase
Induction and activation of tissue transglutaminase part of
apoptotic program & an effector of the death process
Catalyses Ca
2+
-dependent cross link between glutamate &
lysine and production of high molecular mass e(g-
glutamyl)-lysine linked protein polymers
Useful for detection of apoptosis in vivo; apoptosis
initiated in cell
Measured by ELISA
involuting uterus
Immunohistochemical staining for TG
Chromatin Fragmented
Chromatin
Agarose gel
electrophoresis
DNA fragmentation - biochemical
hallmark of apoptosis
DNA cleaved into non-random fragments
180-200 bp fragments & multiples of this unit
Flow cytometry
Light scatter - interaction of cell with light of the laser
beam
Scattered light: provides information about cell size &
structure
Cell size correlates with the intensity of scattered light in
forward direction
Cell granularity, refractivity, ability of intracellular
structures to reflect light correlates with intensity of
scattered light at a right angle (90o) to the laser beam
(side scatter)
Morphological assessment
Flow cytometry detection of changes in cell size and
granularity
Side scatter shows a decrease in granularity
Forward scatter shows a decrease in cell size
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Annexin V - specific apoptosis probe
Based on observation that phosphatidylserine is
translocated to outside leaflet of the plasma membrane
during apoptosis
Annexin V preferentially binds PS
As PS translocates to outer leaflet of the plasma
membrane, time dependent increase in annexin
Coupled to FITC fluorescence
Visible green
Necrosis
Propidiumiodide labels cellular DNA of necrotic cells which
have damaged membrane visible red
Good cells exclude propidiumiodide