Intercalated BSc 2007-08
CELL DEATH
an overview
Dr Cathy Baker
22nd October 2007
How do cells die?
Killed by injurious agents
NECROSIS
Induced to commit suicide
APOPTOSIS
LEARNING OBJECTIVES
Understand, describe and illustrate …
Differences: necrosis vs. apoptosis
Morphological changes of apoptosis
Function of apoptosis
Principal biochemical mechanisms
Role of apoptosis in pathologies
Lecture overview
Necrosis
Function
Apoptosis
Morphological
changes
Biochemistry
Pathology
Necrosis
Mechanical injury & toxic agents
Cell groups
Membrane integrity destroyed
Cells and organelles swell, burst
and leak contents
Inflammatory response
Other cells and tissues damaged
Cell death by necrosis
John Kerr et al
Br.J.Cancer 26: 239-257, 1972
Apoptosis
Essential biological process
Cells have role in own death told or decide to commit suicide
Programmed cell death (PCD)
Apoptosis
Distinct form of single cell death
Tightly regulated
Very localised
Energy consuming process
Membranes intact (early stages)
Safe disposal of cell corpse
No inflammation
Necrosis
Apoptosis
Morphological
changes
Changes in cell morphology
Cells shrink and become detached
from adjoining cells
Cytoskeleton collapses
Mitochondria remain intact
Plasma membrane develops bubbles
(blebs) on surface
Membrane blebs during apoptosis
Nucleus and chromatin condense
Aggregates at periphery of nucleus
Nuclear envelope disintegrates
DNA fragmentation
Budding off and breakage into small
membrane wrapped fragments apoptotic bodies
Formation of apoptotic bodies
What happens to apoptotic cells
and apoptotic bodies?
Ingested & degraded by phagocytes
Macrophages and dendritic cells
Adjacent cells in tissue
High speed and efficiency
Histologically inconspicuous
No inflammation
Phagocytosis of apoptotic cells and bodies
Necrosis
Function
Apoptosis
Morphological
changes
Function of apoptosis?
Deliberate removal of specific,
unwanted cells
Organised and controlled manner
Without damaging other cells or
tissues
Circumstances?
Homeostasis
Constancy of internal environment
Tissue turnover
Cell numbers have to be maintained
Homeodynamics
Embryonic development
Removal of unwanted cells
Damage
Organ and tissue differentiation
Vestigial structures
Alteration of tissue form
5 weeks
8 weeks
Neurological development
Deletion of excess immature
neurons that have failed to establish
synaptic connections
Occurs in CNS and PNS
Prevents redundant cell in mature
nervous system
Involution of tissue
Endometrial breakdown prior to
menstruation
Regression of lactating breast tissue
after weaning
Cell damage
Internal cell damage
Inappropriate 3o protein structure
Cell Infection
Viral
Stress
Starvation
DNA damage
Ionizing radiation, ROS
Necrosis
Function
Apoptosis
Morphological
changes
Biochemistry
Biochemistry of apoptosis
Intense area of research
Complicated integrated mechanisms
Much more to be revealed!
Common core process
Underpins morphological changes
Four stage process
Stage 1 - The Death Signal
Stage 2 - Integration and Transduction
Stage 3 - Execution
Stage 4 - Cell Removal
Stage 1- The Death Signal
Absence or withdrawal of positive
survival factors
Presence of negative pro-apoptotic
factors
Survival or positive signals
Cell survival relies receiving
positive stimuli
Neuronal growth factor
Interleukin 2 for lymphocytes
Hormones
Withdrawal is a death signal
Default pathway for many cells
Death or negative signals
Signals to induce apoptosis
Damaged DNA
UV light and X rays
Chemotherapeutic drugs
Oxidants/free radicals
Oxidative stress
Death activators or receptor
ligands
What are Death Activators?
Molecules that bind to specific
receptors on cell surface
Tumour necrosis factor alpha
Lymphotoxin TNF beta
Fas ligand (CD95)
Binding of death activator to its
specific receptor is a pro-apoptotic
signal
Stage 2 - Integration and Transduction
Signals linked to execution phase
through an integration stage
Uses positive and negative
regulatory molecules
Inhibit, stimulate or forestall
apoptosis
To die or not to die?
Integrated balance between positive
survival factors and negative death
signals decides fate of cell
Common intracellular machinery
for apoptosis
The three main players
 Family of enzymes - Caspases
 Protein family - Bcl-2 proteins
 Regulating gene - p53 gene
Caspases
Family of protease enzymes
14 isoforms identified
Have Cysteine at active site
Synthesised as inactive precursors procaspase
Not all involved in apoptosis
Procaspase structure
prodomain
large subunits
small subunits
cleavage
sites
Procaspase are activated through cleavage
Re-association of large and small subunits
Activated caspase has proteolytic activity
Initiator
caspases
Effector
caspases
Initiator
caspases
Effector
caspases
• Activate other caspases
• Amplify caspase activity
Apoptosis
execution
Bcl-2 proteins
 Large family of proteins
 Named from B cell lymphoma
 Some are pro-apoptotic some are
anti-apoptotic
Bcl-2 proteins and apoptosis
 Main mechanism is regulation of
mitochondrial permeability
 Cell survival stimuli induce the
expression of anti-apoptotic Bcl
proteins
 Death signals induce pro-apoptotic
Bcl proteins
p53 gene and p53 protein
p53 is tumour suppressor gene
Active gene product p53 produced in
response to DNA and cell damage
Prevents cell completing cell cycle
If damage is minor - allows repair
If major - induces apoptosis
Complex mechanisms
Apoptotic transduction pathways
Mitochondrial or intrinsic pathway
Death activator or extrinsic pathway
Intrinsic or mitochondrial pathway
Cell and DNA
damage –
Active p53
Bcl-2
Bax
●Changes in trans-membrane potential
●Pores form in (outer) membrane
●Inner & outer membrane proteins involved
Bcl-2
Bax
Irreversible
cell death
Bcl-2
Bax
Cyt C
Cyt C
Apaf-1
Apoptosis
activating factor -1
Aggregation of Cyt C/Apaf 1 complexes
Binding of Procaspase - 9
ATP
Auto-activation of Procaspase - 9
ATP
Formation of Active Caspase -9
ADP
Death receptor or extrinsic
pathway
Molecules that bind to specific
receptors on cell surface
Tumour necrosis factor alpha (TNF)
Lymphotoxin TNF beta
Fas ligand (CD95)
Binding of death activator to specific
receptor is pro-apoptotic signal caspase activation
Cell membrane with specific death receptors
Binding sites for death activators
Death domains extending into cytosol
Death receptors bind Death Activators
Clustering of death domains
Binding of adaptor protein(s)
Binding of caspase-8
Release of activated caspase-8
3. Execution
Achieved through activation and
deactivation of target proteins
by effector caspases
Activated effector caspases lead to …
Digestion of cytoskeleton proteins
Nucleus and chromatin degradation
Plasma membrane changes
Cytoskeleton degradation
Chromatin
degradation
Caspase-9 enlarges nuclear
pores
Allows entry of Caspase-3 and 7
Activation of nucleases
Caspase Activated DNAase - CAD
ICAD
CAD
Nucleosome
cleavage
CAD
Linker DNA
Nucleosome bead
8 histone molecules +
146 nucleotide pairs of DNA
mw ladder
DNA from
apoptotic cell
Other nuclear
changes
Structural proteins - Lamins
degraded by caspase-6
DNA repair enzymes inactivated
Nuclear membrane degraded
4. Cell removal
What is the eat me signal?
Enzyme system keeps PS on
inner surface
Inhibited during apoptosis
PS redistributed to extra-cellular
surface
Macrophage receptors recognise
and bind PS
Phagocytosis of apoptosome
Release of anti-inflammatory
substances
Necrosis
Function
Apoptosis
Morphological
changes
Biochemistry
Pathology
Homeostasis
Cell numbers have to be maintained
Cell formation
Cell death
Uncontrolled growth of cells
Insufficient apoptosis
Diseases featuring insufficient
apoptosis
Many cancers
Autoimmune Lymphoproliferative
Syndrome (ALPS)
Excessive apoptosis
Uncontrolled cell loss
Diseases featuring excessive
apoptosis
Neurodegenerative
Parkinson’s disease
Alzheimer's disease
Amyotrophic lateral sclerosis (ALS)
Huntingdon’s disease
Diseases featuring excessive
apoptosis
AIDS
Excessive apoptosis of T helper cells
Ischaemia
Cerebral caused by stroke
Cardiac caused by MI
You should now be able to …
Understand, describe and illustrate …
Differences: necrosis vs. apoptosis
Morphological changes of apoptosis
Function of apoptosis
Principal biochemical mechanisms
Role of apoptosis in pathologies