Cell death

• Cells die by one of two mechanisms – necrosis or

apoptosis
• Two physiologically different processes
– Necrosis – death by injury
Apoptosis and necrosis – Apoptosis – death by suicide
• Apoptosis and necrosis have different characteristics

Necrosis Cytological characteristics of necrosis

• Death by accident
• Associated with nonphysiological circumstances that • Initial swelling of the cell
disrupt cellular homeostasis (eg., ischemia, hypoxia and • Rupture of the plasma
poisoning) membrane
• Necrosis is caused by membrane dissolution (osmotic lysis, • Cytoplasm is spilled to the
shear stress, pore-forming proteins, loss of ATP) extracellular environment
• Necrosis is bad because cellular material (including
degradative enzymes) is released into surrounding tissue
• Affects contiguous groups of cell
• Necrosis usually causes inflammatory reaction

Types of Necrosis Apoptosis

• Coagulation Necrosis • Death by design – genetically programmed cell death
– Seen in infarcted organs, e.g.myocardial infarct • Induced by new gene synthesis, primarily in response to
• Liquefaction Necrosis developmental cues
– Softening of the center of an abscess • Requires new RNA and protein synthesis
• Caseous Necrosis – Inhibitors of transcription or translation prevent
– Cheesy, crumbly appearance, e.g. tuberculosis lesion in apoptosis
the lung • 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 Morphologic changes during apoptosis
• This is not death with the headlights on and the traffic • Membranes become irregular
stopped; rather it is akin to the night-time anatomical • Chromatin becomes condensed and
grave-robbing of old, except that for apoptosis the body segregated
may be dismembered, but is not quite dead • Condensation of cytoplasm
• DNA is fragmented
• Cell is fragmented and phagocytosed

The three phases of apoptosis

Apoptotic cell Growth factor deprivation
Death receptors Genotoxic insults
Chemotherapeutic drugs
ceramide
induction
Bcl2 Apaf1 casp9
point of no return AT H2O2
execution P UV
caspases
ΔΨm

degradation
Chromatin condensation
Membrane blebbing
Cell shrinkage
DNA fragmentation

Why die by apoptosis?

Mechanism of apoptosis apoptosis necrosis

• Internal signals – mitochondrial pathway

• External signals – death receptor pathway
• Apoptosis – inducing factor

Spillage of cell content

Apoptotic bodies engulfed by phagocytes
No spillage of cell content Provoke cytokine secretion

No inflammatory response Inflammatory response

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 APOPTOSIS: morphology

Features of apoptosis vs. necrosis organelle

reduction
Apoptosis Necrosis membrane
blebbing &
• Chromatin condensation • Nuclear swelling changes
• Cell shrinkage • Cell swelling cell
• Preservation of organelles • Disruption of organelles mitochondrial
and cell membranes leakage shrinkage
• Rupture of cell and release
• Rapid engulfment by of cellular contents nuclear chromatin
neighboring cells • Inflammatory response fragmentation condensation
preventing inflammation
• Biochemical hallmark -
DNA fragmentation

Blebbing & Apoptotic bodies

Morphological features of apoptosis Bleb

The control retained over the cell
membrane & cytoskeleton allows intact
pieces of the cell to separate for
recognition & phagocytosis

Apoptotic body

Membrane blebbing

DNA fragmentation and gel Other morphological features of

electrophoresis apoptosis
• Digestion of DNA starts after 2 hrs
• 3&4 hrs after initiation of apoptosis
DNA is almost all degraded
Nuclear
• DNA is fragmented with restriction
breakdown
endonucleases
(Hoechst)
• Apoptosis induces 180 bp laddering
of DNA
• Targets of endonuclease attack -
linker regions between nucleosomes

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 Macrophage recognition of the
apoptotic cell Engulfment signals
Call in the macrophages with a surface marker
membrane changes

phosphatidylethanolamine phosphatidylserine phosphatidylcholine sphingomyelin

Apoptotic body
Phosphatidylserine
externalisation
MΦ
Plasma membrane changes:
Phosphatidylserine is exposed externally Detection
Membrane proteins lose their normally Annexin V binding
MΦ the membrane
asymmetric distribution across

Necrosis Necrosis
• The disorganization is primarily Lysosomal spillage
of the plasma & organelle
Cell swelling
membranes because of a power
failure - the energy supply for Mitochondria
the ion pumps Loss of cristae
• Net result - cell swelling & Swelling
lysis/dissolution Disintegration
Chromatin unevenly clumped
ER dilated & dissolved
Ribosomes dispersed
Cytoskeleton eaten up

Apoptosis vs. necrosis Techniques to recognize apoptosis

membranes intact NORMAL
membranes leaky • Morphological assessment
whole cell dissolved
shrinkage • Measurement of tissue transglutaminase
remains controlled spillage
• Measurement of DNA fragmentation
inflammatory
largely ‘nuclear’ • Flow cytometry of apoptotic cells
invites phagocytosis • Specific probes for apoptotic cells – Annexin V

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 Morphological assessment Tissue transglutaminase
control apoptotic
• Induction and activation of tissue transglutaminase part of
apoptotic program & an effector of the death process
• Catalyses Ca2+-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
• Light microscopy initiated in cell
– DNA condensation, nuclear • Measured by ELISA
fragmentation, apoptotic bodies
• Electron microscopy
– Scanning EM
– Transmission EM

DNA fragmentation - biochemical

Immunohistochemical staining for TG hallmark of apoptosis
• DNA cleaved into non-random fragments
• 180-200 bp fragments & multiples of this unit
Agarose gel
electrophoresis

involuting uterus

Chromatin Fragmented
Chromatin

Flow cytometry Morphological assessment

• Light scatter - interaction of cell with light of the laser • Flow cytometry detection of changes in cell size and
beam granularity
• Scattered light: provides information about cell size & – Side scatter – shows a decrease in granularity
structure – Forward scatter – shows a decrease in cell size
• 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)

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 Annexin V - specific apoptosis probe Necrosis
• Based on observation that phosphatidylserine is • Propidium iodide labels cellular DNA of necrotic cells which
translocated to outside leaflet of the plasma membrane have damaged membrane – visible red
during apoptosis • Good cells exclude propidium iodide
• 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