APOPTOSIS

- DR.NILESH CHANDRA

Brief glance at the apoptotic process

Role of apoptosis in healthy physiology, esp. inImmunityProtection of genome

Disruption of apoptosis & its consequences, viz.Neurodegeneartive diseasesCancerChronic inflammatory diseases

Potential therapeutic roles

OBJECTIVES

Apoptosis or programmed cell death, is carefully coordinated collapse of cell, protein degradation, DNA fragmentation followed by rapid engulfment of corpses by neighbouring cells. (Tommi, 2002)

Essential part of life for every multicellular organism from worms to humans. (Faddy et al.,1992)

Apoptosis plays a major role from embryonic development to senescence.

INTRODUCTION

WHY SHOULD A CELL COMMIT SUICIDE?

Apoptosis is needed for proper developmentExamples: The resorption of the tadpole tail The formation of the fingers and toes of the fetus The sloughing off of the inner lining of the uterus The formation of the proper connections between neurons in the brain

Apoptosis is needed for self defense

Examples: Cells infected with viruses Cells of the immune system Cells with DNA damage Cancer cells

WHAT MAKES A CELL DECIDE TO COMMIT SUICIDE?

Withdrawal of positive signalsexamples : growth factors for neurons Interleukin-2 (IL-2)

Receipt of negative signals examples : increased levels of oxidants within the cell damage to DNA by oxidants death activators :

Tumor necrosis factor alpha (TNF-) Lymphotoxin (TNF-β) Fas ligand (FasL)

Cell death by injury

-Mechanical damage -Exposure to toxic chemicals

Cell death by suicide

-Internal signals-External signals

CAUSES OF CELL DEATH:

HISTORY OF CELL DEATH / APOPTOSIS RESEARCH

1800s Numerous observation of cell death1908 Mechnikov wins Nobel prize (phagocytosis) 1930-40 Studies of metamorphosis1948-49 Cell death in chick limb & exploration of NGF1955 Beginning of studies of lysomes1964-66 Necrosis & PCD described1971 Term apoptosis coined1977 Cell death genes in C. elegans1980-82 DNA ladder observed & ced-3 identified1989-91 Apoptosis genes identified, including bcl-2,

fas/apo1 p53, ced-3 sequenced(Richerd et.al., 2001)

NECROSIS VS. APOPTOSIS

Cellular condensation Membranes remain intact Requires ATP Cell is phagocytosed, no

tissue reaction Ladder-like DNA

fragmentation In vivo, individual cells

appear affected

• Cellular swelling

• Membranes are broken

• ATP is depleted

• Cell lyses, eliciting an inflammatory reaction

• DNA fragmentation is random, or smeared

• In vivo, whole areas of the tissue are affected

Necrosis Apoptosis

NECROSIS VS APOPTOSIS

Wilde, 1999

STAGES OF APOPTOSIS

Sherman et al., 1997

Induction of apoptosis related genes, signal transduction

membrane blebbing & changes

mitochondrial leakage

organelle

reduction

cell

shrinkage

nuclear fragmentation

chromatin condensation

APOPTOSIS: Morphology

Hacker., 2000

membrane blebbing & changes

mitochondrial leakage

organelle reduction

cell shrinkage

nuclear fragmentationchromatin condensation

APOPTOSIS: Morphological events

Bleb

Blebbing & Apoptotic bodies

The control retained over the cell membrane & cytoskeleton allows intact pieces of the cell to separate for recognition & phagocytosis by Ms

Apoptotic body

M M

APOPTOSIS: PATHWAYS

Death Ligands

Effector Caspase 3

Death Receptors

Initiator Caspase 8

PCD

DNA damage & p53

Mitochondria/Cytochrome C

Initiator Caspase 9

“Extrinsic Pathway”

“Intrinsic Pathway”

MAJOR PLAYERS IN APOPTOSIS

• Caspases

• Adaptor proteins

• TNF & TNFR family

• Bcl-2 family

LIGAND-INDUCED CELL DEATH

Ligand ReceptorFasL Fas (CD95)TNF TNF-RTRAIL DR4 (Trail-R)

LIGAND-INDUCED CELL DEATH

“The death receptors”

Ligand-induced trimerization

Death Domains

Death Effectors

Induced proximity of Caspase 8

Activation of Caspase 8

FasL

Trail

TNF

p53

Apoptosis events

Initiator caspases 6, 8, 9,12

Activators of initiator enzymes

Apoptotic signals

Execution caspases 2, 3, 7

APOPTOSIS: Signaling & Control pathways I

Externally driven

Internally driven

Cytochrome C

Externally driven

Activation

mitochondrion

p53

ExternalInternal

Apoptosis events

Initiator caspases 6, 8, 9,12

Activators of initiator enzymes

Apoptotic signals

Execution caspases 2, 3, 7 Inhibitors of

apoptosis

APOPTOSIS: Signaling & Control pathways II

Inhibitors

Externally driven

Internally driven

Cytochrome C

Externally driven

Survival factors

Bcl2

Inhibition

H2O2

Growth factorreceptors

casp9Bcl2

PI3KAkt

BAD

Apaf1

Cyt.CATP

The mitochondrial pathway

casp3

casp3

IAPs

Smac/DIABLO

AIF

Bax

Bax

p53

Fas

Casp8

Bid

Bid

Bid

DNA damage

Pollack etal., 2001

Importance of Apoptosis

• Important in normal physiology / development– Development:

– Immune system maturation– Morphogenesis– Neural development

– Adult: – Immune privilege– DNA Damage – Wound repair.

Immune system maturation:

Positive selection of thymocytes in the

thymus. Thymic selection involves

thymic stromal cells (epithelial cells,

dendritic cells, and macrophages), and

results in mature T cells that are both

self-MHC restricted and self-tolerant.

Immune system maturation:

Negative selection of thymocytes in the

thymus. Thymic selection involves

thymic stromal cells (epithelial cells,

dendritic cells, and macrophages), and

results in mature T cells that are both

self-MHC restricted and self-tolerant.

Morphogenesis:

Molecular basis: Morphogens Transcription factors Cell adhesion molecules

Cellular basis: Cell-cell adhesion Cell contractility Extracellular matrix Apoptosis

Immune Privilege:

Fas-ligand (FasL; also called CD95L or Apo-1L)

required for tissues to display a privileged status

FasL functions to induce apoptotic cell death in

most cells that express its receptor, Fas.

Fas-bearing cells include cells of the immune

system

Tissues that naturally express FasL kill

infiltrating lymphocytes and inflammatory

cells.

DNA Damage

Transcriptional Up-Regulation of Target Genes

p21(CDK inhibitor)

GADD45(DNA repair)

BAX(apoptosis gene)

Ionizing Radiation, Carcinogens & Mutagens

DNA Damage

p53 activated and binds to DNA

G1 Arrest

Successful repair

Repair fails

APOPTOSIS

Role of apoptosisIn maintaining

Integrity of genomicDNA in normal cells

Disruption of apoptosis

Two major ways: Inappropriate activation of the

apoptotic process Immune defect in AIDSNeurodegenerative diseases.

Inadequate apoptosisCancerChronic inflammatory conditionsAutoimmune diseases.

Immune defect in AIDS

Profound reduction in the population size of CD4 + T helper cells

Caused by excessive apoptosis

Process includes transfer of regulatory viral gene products (such as HIV-1 Tat) from HIV infected cells to bystander T cells

Renders them susceptible to T cell receptor-induced, CD95-mediated apoptosis.

Neurodegenerative Disease

Apoptosis triggered by amyloid β neurotoxic abnormal protein structures or

aggregates In adult neurodegenerative diseases including

Alzheimer's Huntington's chorea Parkinson's disease, Amyotrophic lateral sclerosis

Amyloid β can exert neurotoxic effects by generation of intracellular oxidative stress increases in calcium ions

Both of these can trigger apoptosis in susceptible cell types.

Cancer

Mutations affect the control mechanisms of apoptosis and cell survival .

Bcl-2 in follicular lymphoma increased bcl-2 expression confers resistance to

chemotherapy in ALL and some forms of AML Bcl-2 blocks the endonucleolytic cleavage of DNA

that is so characteristic of apoptosis. BCR-ABL in CML

Inappropriately prolongs cell survival by inhibiting apoptosis

Recent evidence indicates that BCR-ABL can mimic the modularity signals provided by some cytokines involved in apoptosis.

Chronic Inflammatory conditions:

Intact neutrophils are engulfed by macrophages at the sites of inflammation.

Morphological changes and a chromatin fragmentation pattern, characteristic of apoptosis, within the neutrophils triggers recognition by the macrophages.

Rheumatoid arthritis may reflect prolonged survival of leucocytes that are normally programmed to die by apoptosis.

Therapeutic Significance:

Approaches to counter inappropriate apoptosis:Caspases

are critical to the control of apoptosis several pharmaceutical companies are developing

potent and specific caspase inhibitors have shown great promise in murine models of

inappropriate neuronal apoptosis.

The treatment of certain lymphomas by antisense oligonucleotides to bcl-2

Death-inducing cytokines of the tumour necrosis factor family, such as TRAIL

SUMMARY:

Apoptosis: mechanism & regulation

Importance of apoptosis during early development

Importance of apoptosis during adulthood

Disruption of apoptotic pathway

Therapeutics

References:

Why is apoptosis important to clinicians; Haslett C; BMJ. 2001 June 23; 322(7301): 1499–1500.

Association of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand with Total and Cardiovascular Mortality in Older Adults; Stefano V et al; Atherosclerosis. 2011 April ; 215(2): 452–458. doi:10.1016/j.atherosclerosis.2010.11.004.

Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics; Kerr JF, Wyllie AH, Currie AR; Br J Cancer. Aug;26(4):239-57

Pathologic Basis of Disease; Robbins & Cotran Immunology, 5th Ed; Kuby

DNA DAMAGE

p53

The bcl-2 family

BH4 BH3 BH1 BH2 TMN C

Receptor domain

phosphorylation

Raf-1calcineurin Pore

formation

Membraneanchor

Liganddomain

Group I

Group II

Group III

Bcl-2

bax

Badbidbik

Back

P53 & Apoptosis

p53 first arrests cell growth between G1 S

This allows for DNA repair during delay

If the damage is too extensive then p53 induces gene activation leading to apoptosis (programmed cell death)

BACK

3 mechanisms of caspase activationa. Proteolytic cleavage e.g.

pro-caspase 3

b. Induced proximity, e.g. pro-caspase 8

c. Oligomerization, e.g. cyt c, Apaf-1 & caspase 9

Back

Cytolytic lymphocyte/CTL (& natural killer lymphocyte) presents Fas ligand/CD178 on its surface to tell the infected cell to die

Apoptosis events

Initiator caspases

Apoptotic signals

Execution caspases

Externally driven

Cytochrome c

Fas ligand

Apoptosis signal to kill infected cells

CTL Virally infected cell

Fas/ CD95 is the ‘death receptor’

The immunological synapse holds the cells much tighter together than shown here