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Intercalated BSc 2007-08. CELL DEATH an overview. Dr Cathy Baker 22 nd October 2007. How do cells die?. Killed by injurious agents Induced to commit suicide. NECROSIS. APOPTOSIS. LEARNING OBJECTIVES. Understand, describe and illustrate … Differences: necrosis vs. apoptosis

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Intercalated BSc 2007-08

CELL DEATHan overview

Dr Cathy Baker

22nd October 2007


How do cells die?

  • Killed by injurious agents

  • Induced to commit suicide

NECROSIS

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


Necrosis

Apoptosis

Lecture overview

Function

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


Neurologicaldevelopment

  • 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


cleavage

sites

prodomain

large subunits

small subunits

Procaspase structure


Procaspase are activated through cleavage


Re-association of large and small subunits


Initiator

caspases

Effector

caspases

Activated caspase has proteolytic activity


  • Activate other caspases

  • Amplify caspase activity

Initiator

caspases

Apoptosis

execution

Effector

caspases


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


Cell and DNA

damage –

Active p53

Bax

Intrinsic or mitochondrial pathway

Bcl-2


  • 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

Apoptosis

activating factor -1

Apaf-1


Aggregation of Cyt C/Apaf 1 complexes


Binding of Procaspase - 9

ATP


ATP

Auto-activation of Procaspase - 9


ADP

Formation of Active Caspase -9


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


Binding sites for death activators

Death domains extending into cytosol

Cell membrane with specific death receptors


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


CAD

ICAD

Nucleosome

cleavage

Caspase Activated DNAase - CAD


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


Cell formation

Cell death

Homeostasis

  • Cell numbers have to be maintained


  • 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


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