Cell Death
Normally cells in homeostasis
Physiological and pathological stress
Cellular Adaptation ( Reversible on withdrawal of stimulus)
If the irritant stress persists for long time
Cell injury
Reversible cell injury
Irreversible cell injury (Cell Death) – Apoptosis , Necrosis
Apoptosis
• Definition
• Types
• Mechanisms
• Morphological changes in apoptosis
• Diagnosis in Apoptosis
• Differences from Necrosis
Definition
• Cell suicide
• It is a pathway of cell death that is induced by a tightly regulated
intracellular program in which cells destined to die activate enzymes
( caspase) degrade the cells own nuclear DNA and cytoplasmic proteins.
• The cell is phagoctosed
• There is no leakage outside
• So there is no inflammation
• It is the way of elimination of unwanted cells and those cells which are
damaged beyond repair capacity of cell.
• Apoptosis generally involves single cell in contrast
to necrosis that usually involve a group of cells.
• It is the ‘Programmed cell death’.
• It is genetically programmed.
• It is an energy dependent process.
Types
• Physiological causes
1. Embryogenesis
2. Elimination of potentially self reacting lymphocytes
3. Hormone dependent involution of uterus and breast.
4. Death of cells that have completed their functions.
Pathological Causes
1. Cell death in tumours exposed to chemotherapeutic
agents.
2. Progressive depletion of CD4+ T cells in the pathogenesis
of AIDS.
3. Cell death in viral infections e.g. formation of councilman
bodies in viral hepatitis.
Mechanisms
• Apoptosis can be induced through two distinct but
convergent pathways :-
• Extrinsic Pathway – It is initiated by extracellular
stimulus with help of specific receptors called death
receptors.
• Intrinsic pathway- It is result of increased
mitochondrial membrane permeability and release
of pro- apoptotic markers like cytochrome C into
the cytoplasm.
Mechanism
Extrinsic pathway Intrinsic pathway
Initiation
Initiation
Caspase 9 Execution
Execution
Caspase 8
Phases
• Apoptosis can be divided into two phases:-
1. Initiation phase : starts with stimulus ( Either
extrinsic or intrinsic) and consist of catabolic
activation of caspase like 8 or 9.
2. Execution phase: executioner caspases act to
cause cell death.
Extrinsic pathway Initiation phase
• It is initiated through specific receptor called death
receptors.
• Fas protein ( CD95)
• TNF receptors
Fas protein (CD95)
Fas receptor ( Death receptor)
Multiple Fas proteins come together
Cytoplasmic death domains combine to form a death domain ( FADD)
Activate pro-caspasae 8 to activate caspase 8
TNF
TNF receptor
Multiple Fas protein come together
Cytoplasmic death domains combine to form a death domain TRADD
Activate pro-caspase 8 to activate caspase 8
Intrinsic pathway Initiation phase
• The intrinsic signaling pathways that initiate
apoptosis involve a diverse array of non-receptor-
mediated stimuli that produce intracellular signals
within the cell and are mitochondrial-initiated
events.
• Stimuli
• DNA damage ( beyond repair)
• DNA damage can occur after exposure to agents like
radiation and chemotherapy ( genotoxic stress)
Stimuli
Anti apoptotic molecules Bcl-2 and Bcl-x are lost
Replaced by pro-apoptotic molecules like Bak, Bax, Bim
Increased mitochondrial permiability (MPT)
Release to cytochrome C into cytoplasm
Activates Apaf -1 along with procaspase – 9
Activated caspase-9
• Antiapoptotic molecules
1. BCL-2
2. BCL-X
3. Mcl-1
4. FLIP
• Proapoptotic molecules
1. Bax
2. Bim
3. Bad
4. Bak
5. P53 gene
Extrinsic pathway Intrinsic pathway
caspase9
Caspase 8
Activates caspase 3 and 7
Sequentially activates all other caspase
Caspase cleave cytoskeletal and nuclear matrix protein
Cell degenerate/ apoptotic bodies formed
Phagocytosis
Phosphatidylserine is a phospholipid present on inner surface membrane normally
During Apoptosis
Flipped to outer surface
Externalization of phosphatidylserine causes its tagging
Phagocytes ingulf it
• These alterations permit the early recognition of
apoptotic cells by macrophages
• Phagocytosis without the release of proinflammatory cellular
components.
• The process of apoptotic cells is so efficient that dead cells
disappear without leaving a trace and inflammation is
virtually absent.
• Essential feature is immediate, specific and non-
inflammatory nature of phagocytosis
Morphological Changes in Apoptosis
1. Cell shrinkage is earliest change ( it is due to
damage to cytoskeletal proteins).
2. Cellular organelles are tightly packed thus
imparting intense eosinophilic color to cytoplasm.
3. Nuclear changes
• Pyknosis i.e Chromatin condensation or nuclear
compaction
• Karyorrhexis i.e nuclear fragmentation .It is due to
activity of endonuclease and caspases
• It is the most characteristic feature.
Morphological Changes in Apoptosis
4. Cytoplasmic membrane shows multiple surface
blebs.
• It is the end stage of apoptosis
• Cell membrane intact thus preventing leaking out of cellular
contents.
5. Nuclear fragments and cytoplasmic organelles form
membrane bound apoptotic bodies.
These are membrane bound round masses of eosinophilic
cytoplasm with tightly packed organelles which may contain
nuclear debris.
• These apoptotic bodies are recognised by
phagocytes and destroyed.
• Characteristically unlike necrosis there is no acute
inflammatory reaction around apoptosis.
Diagnosis of Apoptosis
• Annexin- V is a recombinant protein with high
affinity for phospholipid like phosphatidylserine.
• Phosphatidylserine is a phospholipid present on inner
surface membrane normally but it is flipped to outer surface
during apoptosis thus become a marker of apoptosis.
Agarose gel electrophoresis
• Fragmented DNA shows Step ladder pattern which is due to
internucleosomal cleavage of DNA by endonuclease.
• During karryorrhexis endonuclease activation leaves short
DNA fragments regularly spaced in size.
• This laddered pattern is characteristic but not specific for
apoptosis.
