apoptosis seminar

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apoptosis; physiological importance; mechanism in brief; clinical applications


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Brief glance at the apoptotic process Role of apoptosis in healthy physiology, esp. in Immunity Protection of genome Disruption of apoptosis & its consequences, viz. Neurodegeneartive diseases Cancer Chronic 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 development Examples: 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?:

What makes a cell decide to commit suicide? Withdrawal of positive signals examples : 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 )

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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:

History of cell death / apoptosis research 1800s Numerous observation of cell death 1908 Mechnikov wins Nobel prize (phagocytosis) 1930-40 Studies of metamorphosis 1948-49 Cell death in chick limb & exploration of NGF 1955 Beginning of studies of lysomes 1964-66 Necrosis & PCD described 1971 Term apoptosis coined 1977 Cell death genes in C. elegans 1980-82 DNA ladder observed & ced-3 identified 1989-91 Apoptosis genes identified, including bcl-2, fas/apo1 & p53, ced-3 sequenced (Richerd et.al., 2001)

Necrosis vs. Apoptosis:

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




STAGES OF APOPTOSIS Sherman et al., 1997 Induction of apoptosis related genes, signal transduction

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membrane blebbing & changes mitochondrial leakage organelle reduction cell shrinkage nuclear fragmentation chromatin condensation APOPTOSIS: Morphology Hacker., 2000

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membrane blebbing & changes mitochondrial leakage organelle reduction cell shrinkage nuclear fragmentation chromatin condensation APOPTOSIS: Morphological events

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Bleb Blebbing & Apoptotic bodies The control retained over the cell membrane & cytoskeleton allows intact pieces of the cell to separate for recognition & phagocytosis by M F s Apoptotic body M F M F

Apoptosis: Pathways:

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”

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MAJOR PLAYERS IN APOPTOSIS Caspases Adaptor proteins TNF & TNFR family Bcl-2 family

Ligand-induced cell death:

Ligand-induced cell death Ligand Receptor FasL Fas (CD95) TNF TNF-R TRAIL DR4 (Trail-R)

Ligand-induced cell death:

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

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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

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p53 External Internal 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

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H 2 O 2 Growth factor receptors casp9 Bcl2 PI3K Akt BAD Apaf1 Cyt.C ATP The mitochondrial pathway casp3 casp3 IAPs Smac/ DIABLO AIF Bax Bax p53 Fas Casp8 Bid Bid Bid DNA damage Pollack etal ., 2001

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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::

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::

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::

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:

DNA Damage Ionizing Radiation, Carcinogens & Mutagens DNA Damage p53 activated and binds to DNA G1 Arrest Successful repair Repair fails APOPTOSIS Role of apoptosis In maintaining Integrity of genomic DNA in normal cells

Disruption of apoptosis:

Disruption of apoptosis Two major ways: Inappropriate activation of the apoptotic process Immune defect in AIDS Neurodegenerative diseases. Inadequate apoptosis Cancer Chronic inflammatory conditions Autoimmune diseases.

Immune defect in AIDS:

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:

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::

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::

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, 5 th Ed; Kuby

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The bcl-2 family BH4 BH3 BH1 BH2 TM N C Receptor domain phosphorylation Raf-1 calcineurin Pore formation Membrane anchor Ligand domain Group I Group II Group III Bcl-2 bax Bad bid bik Back

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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

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3 mechanisms of caspase activation a. 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

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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

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