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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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How do cells die

Intercalated BSc 2007-08

CELL DEATHan overview

Dr Cathy Baker

22nd October 2007


How do cells die

How do cells die?

  • Killed by injurious agents

  • Induced to commit suicide

NECROSIS

APOPTOSIS


Learning objectives

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


How do cells die

Necrosis

Apoptosis

Lecture overview

Function

Morphological

changes

Biochemistry

Pathology


Necrosis

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


How do cells die

Cell death by necrosis


How do cells die

John Kerr et al

Br.J.Cancer 26: 239-257, 1972


Apoptosis

Apoptosis

  • Essential biological process

  • Cells have role in own death - told or decide to commit suicide

  • Programmed cell death (PCD)


Apoptosis1

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


How do cells die

Necrosis

Apoptosis

Morphological

changes


Changes in cell morphology

Changes in cell morphology

  • Cells shrink and become detached from adjoining cells

  • Cytoskeleton collapses

  • Mitochondria remain intact

  • Plasma membrane develops bubbles (blebs) on surface


How do cells die

Membrane blebs during apoptosis


How do cells die

  • 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


How do cells die

Formation of apoptotic bodies


What happens to apoptotic cells and 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


How do cells die

Phagocytosis of apoptotic cells and bodies


How do cells die

Necrosis

Function

Apoptosis

Morphological

changes


Function of apoptosis

Function of apoptosis?

  • Deliberate removal of specific, unwanted cells

  • Organised and controlled manner

  • Without damaging other cells or tissues

Circumstances?


Homeostasis

Homeostasis

  • Constancy of internal environment

  • Tissue turnover

  • Cell numbers have to be maintained

Homeodynamics


How do cells die

Embryonic development

Removal of unwanted cells

  • Damage

  • Organ and tissue differentiation

  • Vestigial structures

  • Alteration of tissue form


How do cells die

5 weeks

8 weeks


How do cells die

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


How do cells die

Involution of tissue

  • Endometrial breakdown prior to menstruation

  • Regression of lactating breast tissue after weaning


How do cells die

Cell damage

  • Internal cell damage

    • Inappropriate 3o protein structure

  • Cell Infection

    • Viral

  • Stress

    • Starvation

  • DNA damage

    • Ionizing radiation, ROS


How do cells die

Necrosis

Function

Apoptosis

Morphological

changes

Biochemistry


Biochemistry of apoptosis

Biochemistry of apoptosis

  • Intense area of research

  • Complicated integrated mechanisms

  • Much more to be revealed!

  • Common core process

  • Underpins morphological changes

  • Four stage process


How do cells die

Stage 1 - The Death Signal


How do cells die

Stage 2 - Integration and Transduction


How do cells die

Stage 3 - Execution


How do cells die

Stage 4 - Cell Removal


How do cells die

Stage 1- The Death Signal

  • Absence or withdrawal of positive survival factors

  • Presence of negative pro-apoptotic factors


Survival or positive signals

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

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

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

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

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

Common intracellular machinery for apoptosis

The three main players

  • Family of enzymes - Caspases

  • Protein family - Bcl-2 proteins

  • Regulating gene - p53 gene


Caspases

Caspases

  • Family of protease enzymes

  • 14 isoforms identified

  • Have Cysteine at active site

  • Synthesised as inactive precursors - procaspase

  • Not all involved in apoptosis


How do cells die

cleavage

sites

prodomain

large subunits

small subunits

Procaspase structure


How do cells die

Procaspase are activated through cleavage


How do cells die

Re-association of large and small subunits


How do cells die

Initiator

caspases

Effector

caspases

Activated caspase has proteolytic activity


How do cells die

  • Activate other caspases

  • Amplify caspase activity

Initiator

caspases

Apoptosis

execution

Effector

caspases


Bcl 2 proteins

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

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 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

Apoptotic transduction pathways

  • Mitochondrial or intrinsic pathway

  • Death activator or extrinsic pathway


Intrinsic or mitochondrial pathway

Cell and DNA

damage –

Active p53

Bax

Intrinsic or mitochondrial pathway

Bcl-2


How do cells die

  • Changes in trans-membrane potential

  • Pores form in (outer) membrane

  • Inner & outer membrane proteins involved

Bcl-2

Bax


How do cells die

Irreversible

cell death

Bcl-2

Bax

Cyt C


How do cells die

Cyt C

Apoptosis

activating factor -1

Apaf-1


How do cells die

Aggregation of Cyt C/Apaf 1 complexes


How do cells die

Binding of Procaspase - 9

ATP


How do cells die

ATP

Auto-activation of Procaspase - 9


How do cells die

ADP

Formation of Active Caspase -9


Death receptor or extrinsic pathway

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


How do cells die

Binding sites for death activators

Death domains extending into cytosol

Cell membrane with specific death receptors


How do cells die

Death receptors bind Death Activators


How do cells die

Clustering of death domains


How do cells die

Binding of adaptor protein(s)


How do cells die

Binding of caspase-8


How do cells die

Release of activated caspase-8


3 execution

3. Execution

  • Achieved through activation and deactivation of target proteins by effector caspases


Activated effector caspases lead to

Activated effector caspases lead to …

  • Digestion of cytoskeleton proteins

  • Nucleus and chromatin degradation

  • Plasma membrane changes


How do cells die

Cytoskeleton degradation


Chromatin degradation

Chromatin degradation

  • Caspase-9 enlarges nuclear pores

  • Allows entry of Caspase-3 and 7

  • Activation of nucleases


How do cells die

CAD

ICAD

Nucleosome

cleavage

Caspase Activated DNAase - CAD


How do cells die

CAD

Linker DNA

Nucleosome bead

8 histone molecules +

146 nucleotide pairs of DNA


How do cells die

mw ladder

DNA from

apoptotic cell


Other nuclear changes

Other nuclear changes

  • Structural proteins - Lamins degraded by caspase-6

  • DNA repair enzymes inactivated

  • Nuclear membrane degraded


How do cells die

4. Cell removal

What is the eat me signal?


How do cells die

  • 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


How do cells die

Necrosis

Function

Apoptosis

Morphological

changes

Biochemistry

Pathology


Homeostasis1

Cell formation

Cell death

Homeostasis

  • Cell numbers have to be maintained


How do cells die

  • Uncontrolled growth of cells

  • Insufficient apoptosis


Diseases featuring insufficient apoptosis

Diseases featuring insufficient apoptosis

  • Many cancers

  • Autoimmune Lymphoproliferative Syndrome (ALPS)


How do cells die

  • Excessive apoptosis

  • Uncontrolled cell loss


Diseases featuring excessive apoptosis

Diseases featuring excessive apoptosis

  • Neurodegenerative

    • Parkinson’s disease

    • Alzheimer's disease

    • Amyotrophic lateral sclerosis (ALS)

    • Huntingdon’s disease


Diseases featuring excessive apoptosis1

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

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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