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Apoptosis Apoptosis Dr.CSBR.Prasad, M.D.

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Cell injuryCell injury• Reversible injuryReversible injury 1-Cell swelling 2-Fatty change 3-Mitochondrial swelling 4-ER disruption 5-Membrane blebs 6-Cytoskeletal disruption• Irreversible injuryIrreversible injury 1-Apoptosis 2-Necrosis

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Examples for irreversible injuryExamples for irreversible injury

1. Apoptosis2. Necrosis

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ApoptosisApoptosis• Initially recognized in 1972 • Greek term meaning “falling offfalling off”• It occurs in many situations (Physiological / Pathological)(Physiological / Pathological)

• It’s a mechanism to eliminate…. –Unwanted cells–Potentially harmful cells –Useless cells –Cells damaged beyond repair

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ApoptosisApoptosisApoptosis is “Programmed cell death”Planned self destruction

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ApoptosisApoptosisClinical implications for this knowledge:

1. Apoptosis is very essential for our survival

2. Increased or decreased apoptosis is associated with diseases

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ApoptosisApoptosisApoptosis in Physiologic Situations: • Destruction of cells during embryogenesis • Hormone-dependent involution in the adult • Death of host cells that have served their useful

purpose • Elimination of potentially harmful self-reactive

lymphocytes • Cell death induced by cytotoxic T cells

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ApoptosisApoptosis

Apoptosis in Pathological Conditions: • Cell death produced by a variety of injurious

stimuli • Cell injury in certain viral diseases • Pathologic atrophy in parenchymal organs after

duct obstruction• Cell death in tumors

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ApoptosisApoptosis

NOTENOTE::Even in situations in which cell death is

mainly by necrosis, the pathway of apoptosis may contribute

Eg: Injurious stimuli that cause increased mitochondrial permeability trigger apoptosis

Activation of caspases Activation of caspases is the is the basisbasis for for

apoptosisapoptosis

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Activation of caspases is the basis for apoptosisActivation of caspases is the basis for apoptosis

• In radiation injury which will be altered, Bax or Bcl2?

• In cancer cells which one you think will be altered Bax or Bcl2?

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ApoptosisApoptosisMorphologyMorphology.

Cell shrinkage. The cell is smaller in size; the cytoplasm is dense; and the organelles are more tightly packed

Chromatin condensation. The chromatin aggregates peripherally, under the nuclear membrane, into dense masses of various shapes and sizes. The nucleus itself may break up, producing two or more fragments.

Formation of cytoplasmic blebs and apoptotic bodies. The apoptotic cell first shows extensive surface blebbing, then undergoes fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and tightly packed organelles, with or without nuclear fragments

Phagocytosis of apoptotic cells or cell bodies, usually by macrophages. The apoptotic bodies are rapidly degraded within lysosomes, and the adjacent healthy cells migrate or proliferate to replace the space occupied by the now deleted apoptotic cell.

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Cytoplasmic blebs

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Apoptosis of epidermal cells in an immune-mediated reaction.

The apoptotic cells are visible in the epidermis with intensely eosinophilic cytoplasm and small, dense nuclei.

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ApoptosisApoptosisBIOCHEMICAL FEATURES OF APOPTOSIS Protein Cleavage: by activation of caspasesDNA Breakdown: DNAses, Endonucleases Phagocytic Recognition: Apoptotic cells express

Phosphatidylserine [Annexin V ]

Thrombospondin other proteins secreted by phagocytes may bind

to apoptotic cells

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Agarose gel electrophoresis of DNA extracted from culture cells

A-Control, B-Extensive apoptosis, C-Cells showing massive necrosis

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ApoptosisApoptosisMECHANISMS OF APOPTOSISMECHANISMS OF APOPTOSIS

There are mainly two mechanismsThere are mainly two mechanisms

1- The extrinsic Pathway (Death Receptor-Initiated)2- The Intrinsic Pathway (Mitochondrial)

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Activation of caspases is the basis for apoptosisActivation of caspases is the basis for apoptosis

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The extrinsic Pathway

(Death Receptor-Initiated)

Fas: death receptor

FasL: Fas Ligand eg: TNF1

FADD: Fas-associated death domain

FLIP: is a naturally occuring preventer of

apoptosis

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The Intrinsic (Mitochondrial) PathwayThe Intrinsic (Mitochondrial) Pathway

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Extrinsic & Mitochondrial Pathways - Extrinsic & Mitochondrial Pathways - SimplifiedSimplified

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EXAMPLES OF APOPTOSISEXAMPLES OF APOPTOSIS

• Apoptosis After Growth Factor Apoptosis After Growth Factor DeprivationDeprivation

• DNA Damage-Mediated ApoptosisDNA Damage-Mediated Apoptosis• Apoptosis Induced by Tumor Necrosis Apoptosis Induced by Tumor Necrosis

Factor Family of ReceptorsFactor Family of Receptors• Cytotoxic T-Lymphocyte-Mediated Cytotoxic T-Lymphocyte-Mediated

ApoptosisApoptosis

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Apoptosis detection methods:

• Caspase-3 is a marker of early apoptosis• Annexin-V

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IHC for TUNEL in murine liver, brown cell is apoptotic.

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Main differences between apoptosis and necrosis

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SDUMC-Path-CSBRPFigure 1-16  Timing of biochemical and morphologic changes in cell injury.

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Differences between Apoptosis and NecrosisDifferences between Apoptosis and Necrosis

Feature Apoptosis NecrosisCell membrane Intact Leaky

Inflammation Absent Present

Cytosolic proteins Normal Altered

Cell death Programmed Unregulated

Leakage of contents No leakage Leakage occurs

Setting Occurs normally also Always pathological

Mechanisms Similarities exist Similarities exist

Number of cells involved Single cell or a few cells Numerous cells

Cell death Final event Initial event

“Fallen leaf sign”Unicameral bone cyst

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E N DE N D

• Falling leaves animation• Chip away the unwanted o give a shape• Dropout necrosis – liver• Effete cells in endometrium• Alzhemer’s disease.• Apoptosis video• Organogenesis• Fallen leaf sign of unicameral bone cyst• Apoptosis is the feature of anoxic brain necrosis in infants• Include the man who described apoptosis

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