CELL DEATHan overview

Dr Cathy Baker

22nd October 2007

Intercalated BSc 2007-08

How do cells die?

Killed by injurious agents

Induced to commit suicide

NECROSIS

APOPTOSIS

LEARNING OBJECTIVES

Understand, describe and illustrate …Differences: necrosis vs. apoptosisMorphological changes of apoptosisFunction of apoptosisPrincipal biochemical mechanisms Role of apoptosis in pathologies

Necrosis

Apoptosis

Pathology

Morphologicalchanges

Function

Biochemistry

Lecture overview

Necrosis

Mechanical injury & toxic agentsCell groupsMembrane integrity destroyedCells and organelles swell, burst

and leak contentsInflammatory responseOther 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 deathTightly regulatedVery localisedEnergy consuming processMembranes intact (early stages)Safe disposal of cell corpseNo inflammation

Necrosis

Apoptosis

Morphologicalchanges

Changes in cell morphology

Cells shrink and become detached from adjoining cells

Cytoskeleton collapsesMitochondria remain intactPlasma 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 phagocytesMacrophages and dendritic cellsAdjacent cells in tissueHigh speed and efficiencyHistologically inconspicuousNo inflammation

Phagocytosis of apoptotic cells and bodies

Necrosis

Apoptosis

Morphologicalchanges

Function

Function of apoptosis?

Deliberate removal of specific, unwanted cells

Organised and controlled manner

Without damaging other cells or tissues

Circumstances?

Homeostasis

Constancy of internal environmentTissue turnoverCell numbers have to be maintained

Homeodynamics

Removal of unwanted cells

Damage

Organ and tissue differentiation

Vestigial structures

Alteration of tissue form

Embryonic development

5 weeks 8 weeks

Deletion of excess immature neurons that have failed to establish synaptic connections

Occurs in CNS and PNS

Prevents redundant cell in mature nervous system

Neurological development

Endometrial breakdown prior to menstruation

Regression of lactating breast tissue after weaning

Involution of tissue

Internal cell damage Inappropriate 3o protein structure

Cell InfectionViral

StressStarvation

DNA damage Ionizing radiation, ROS

Cell damage

Necrosis

Apoptosis

Morphologicalchanges

Function

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

Absence or withdrawal of positive survival factors

Presence of negative pro-apoptotic factors

Stage 1- The Death Signal

Survival or positive signals

Cell survival relies receiving positive stimuli

Withdrawal is a death signalDefault pathway for many cells

Neuronal growth factorInterleukin 2 for lymphocytesHormones

Signals to induce apoptosisDamaged DNA

UV light and X raysChemotherapeutic drugsOxidants/free radicals

Oxidative stressDeath activators or receptor

ligands

Death or negative signals

What are Death Activators?

Molecules that bind to specific receptors on cell surfaceTumour necrosis factor alphaLymphotoxin TNF betaFas 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

CaspasesFamily of protease enzymes

14 isoforms identified

Have Cysteine at active site

Synthesised as inactive precursors - procaspase

Not all involved in apoptosis

prodomain

large subunits

small subunits

cleavagesites

Procaspase structure

Procaspase are activated through cleavage

Re-association of large and small subunits

Activated caspase has proteolytic activity

Initiatorcaspases

Effectorcaspases

Effectorcaspases

Apoptosisexecution

Initiatorcaspases

• Activate other caspases• Amplify caspase activity

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 damagePrevents cell completing cell cycle If damage is minor - allows repairIf major - induces apoptosisComplex mechanisms

Apoptotic transduction pathways

Mitochondrial or intrinsic pathway

Death activator or extrinsic pathway

Intrinsic or mitochondrial pathway

Bcl-2

Bax

Cell and DNAdamage –Active p53

Bax

Bcl-2

●Changes in trans-membrane potential●Pores form in (outer) membrane●Inner & outer membrane proteins involved

Bax

Bcl-2

Cyt C

Irreversiblecell death

Cyt C

Apaf-1 Apoptosisactivating 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 surfaceTumour 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

Nucleosome bead8 histone molecules +

146 nucleotide pairs of DNA

Linker DNA

CAD

mw ladder DNA from apoptotic cell

Other nuclear changes

Structural proteins - Lamins degraded by caspase-6

DNA repair enzymes inactivated

Nuclear membrane degraded

What is the eat me signal?

4. Cell removal

Enzyme system keeps PS on inner surface

Inhibited during apoptosisPS redistributed to extra-cellular

surface Macrophage receptors recognise

and bind PSPhagocytosis of apoptosomeRelease of anti-inflammatory

substances

Necrosis

Apoptosis

Morphologicalchanges

Function

Biochemistry

Pathology

Homeostasis

Cell numbers have to be maintained

Cell formation Cell death

Uncontrolled growth of cellsInsufficient apoptosis

Diseases featuring insufficient apoptosis

Many cancersAutoimmune Lymphoproliferative

Syndrome (ALPS)

Excessive apoptosisUncontrolled cell loss

Diseases featuring excessive apoptosis

NeurodegenerativeParkinson’s diseaseAlzheimer's diseaseAmyotrophic lateral sclerosis (ALS)Huntingdon’s disease

Diseases featuring excessive apoptosis

AIDS Excessive apoptosis of T helper cells

IschaemiaCerebral caused by strokeCardiac caused by MI

You should now be able to …

Understand, describe and illustrate …Differences: necrosis vs. apoptosisMorphological changes of apoptosisFunction of apoptosisPrincipal biochemical mechanisms Role of apoptosis in pathologies