Apoptosis

APOPTOSIS
  • Regulated mechanism of cell death that serves to eliminate unwanted and irreparably damaged cells, with the least possible host reaction.
  1. Apoptosis is self-initiated.
  2. Apoptosis is the hallmark of programmed cell death
  3. Graft versus host disease ,Menstrual cycle,Pathological atrophy following duct obstruction are examples of apoptosis.
MORPHOLOGICAL CHANGES OF APOPTOSIS
  • Cell shrinkage : It is the earliest changes. It is due to damage to cytoskeletal proteins.
  • Chromatin condensation (pyknosis)/nuclear compaction : It is the most characteristic feature.
  • Formation of cytoplasmic blebs : It is the end stage of apoptosis.
  • Cytoplasmic eosinophilia.
  • Chromosomal DNA fragmentation : Internucleosomal cleavage of DNA:It is due to activity of endonuclease and caspases.
  • Formation of apoptotic bodies : These are membrane bound round masses of eosinophilic cytoplasm with tightly packed orgaelles which may contain nuclear debries.
  1. Important examples of apoptotic bodies are civatte bodies, kamino bodies, councilman bodies, Tingible bodies, sunburn cells, satellite dyskeratotic cells, and eosinophilic globules.
  • Phagocytosis of apoptotic cells and bodies by adjacent macrophages or healthy parenchymal cells.
  • Considerable apoptosis may occur before it becomes apparent on histological section.
  • Two very important differentiating features from necrosis are :?
  1. Absence of inflammation.
  2. Intact cell membrane.
PHASES OF APOPTOSIS
INITIATION PHASE

  • Intrinsic (Mitochondrial) Pathway - major mechanism of apoptosis
  1. Mitochondrias are the most important organelles involved in initiation and regulation of apoptosis.
  2. Inducers of apoptosis are growth factor withdrawal, detachment from matrix, glucocorticoids, cytotoxic drugs,DNA damage (by radiation, toxins, free radicals),Protein misfolding (ER stress) and immune cytolysis
  • Pro-apoptotic factors
  1. BAX
  2. BAK
  3. p53
  • Anti-apoptotic factors
  1. BCL-2(BCL-2 is the gene for Apoptosis)
  2. BCL-XL
  3. MCL-1 
  • Sensors of cellular stress and damage
  1. BAD
  2. BIM
  3. BID
  4. Puma
  5. Noxa
  • Cytochrome C has a direct role in Apoptosis.
  • Cytochrome c binds to a protein called APAF-1 (apoptotic protease activating factor-1), which forms apoptosome Caspase-9 - initiator caspase(cysteinyl aspartate specific proteases)
Extrinsic (Death Receptor-Initiated) Pathway

  • Initiated by receptor-ligand interactions
  • Responsible for elimination of self-reactive lymphocytes and damage by cytotoxic T lymphocytes
  • Death receptors (pro-apoptotic)
  • Type 1 TNF receptor (TNFR1)
  • Fas (CD95):CD 95 induces apoptosis when it engaged by fas ligand system.
  • CD 95 is a marker of extrinsic pathway of Apoptosis.
  1. Initiator caspases: caspase-8 and caspase-10.
EXECUTION PHASE:
  • Executioner caspases: caspase-3 and caspase-7
DYSREGULATED APOPTOSIS
  • Defective apoptosis and increased cell survival - autoimmune diseases
  • Increased apoptosis and excessive cell death
  1. Neurodegenerative diseases
  2. Ischemic injury (MI, stroke)
  3. Viral infections
IDENTIFICATION OF APOPTOSIS
  • Staining of chromatin condensation
  • Flow cytometry to visualize rapid cell shrinkage
  • DNA changes detected by in situ techniques or by gel electrophoresis
  • Annexin V Assay, a classical technique for detecting apoptosis, is the most commonly used method for detecting apoptosis by flow cytometry.
  1. Annexin V is a calcium-dependent phospholipid binding protein that has a high affinity for the phophatidylserine (PS), a plasma membrane phospholipid.
  2. One of the earliest features of apoptosis is the translocation of PS from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external
  3. environment.
  4. Annexin V binds to PS exposed on the cell surface and identifies cells at an earlier stage of apoptosis than assays based on DNA fragmentation.
Exam Question of:
  • Inflammation is absent in Apoptosis.
  • Annexin V is a marker of Apoptosis.
  • Cell shrinkage ,Chromosomal breakage, Clumping of chromatin,nuclear condensation and fragmentation ,Intact cell membrane ,Cytoplasmic eosiophilia is seen in Apoptosis
  • Councilman Bodies ,Graft versus host disease ,Menstrual cycle,Pathological atrophy following duct obstruction are examples of apoptosis.
  • Cytochrome C has a direct role in Apoptosis.
  • Memory cells doesn't undergo apoptosis due to presence of Nerve growth factor.
  • Organelle that plays a pivotal role in apoptosis is mitochondria.
  • Apoptosis is inhibited by bcl-2.
  • In apoptosis, Apaf-1 is activated by release of which of Cytochrome c from the mitochondria.
  • The most characteristic feature of apoptosis is condensation of nuclear chromatin which corresponds to nuclear compaction (pyknosis) on light microscopy.
  • Intact cell membrane is also a characteristic feature of Apoptosis .
  • Cysteinyl aspartate specific proteases (Caspases) is involved in Apoptosis.
  • Considerable apoptosis may occur in tissues before it becomes apparent in histology.
  • Apoptotic cells appear round mass of the intensely eosinophilic cytoplasm with dense nuclear chromatin fragments.
  • Macrophages phagocytose the apoptotic cells and degrade them.
  • The normal cellular counterparts of oncogenes are important for inhibition of Apoptosis.
  • Peripheral aggregation of chromatin characterizes Apoptosis.
  • Chemotherapeutic drugs can cause both necrosis and apoptosis.
  • CD 95 is a marker of extrinsic pathway of Apoptosis.
  • Caspases is activated for nuclear fragmentation in apoptosis.
  • Ladder pattern of DNA electrophoresis in apoptosis is caused by the action of endonuclease enzyme.
  • Internucleosomal cleavage of DNA is characteristic of Apoptosis.
  • Phosphatidyl serine has important role in Apoptosis.
  • Starting point of apoptosis for programme cell death is activation of caspases.
  • Apoptosis is self-initiated.
  • Apoptosis is the hallmark of programmed cell death .
  • CD 95 induces apoptosis when it engaged by fas ligand system.
  • BCL-2 is the gene for apoptosis .
  • The lymphocytopenia seen a few hours after administration of a large dose of prednisone to a patient with lymphocytic leukemia is due to massive lymphocytic Apoptosis.
  • Ubiquitin is required for Apoptosis.
  • Inducers of apoptosis are growth factor withdrawal, detachment from matrix, glucocorticoids, cytotoxic drugs and immune cytolysis.
  • The earliest change seen in apoptosis is Cell shrinkage.
  • Execution caspases of apoptosis are Caspase 3 & 7
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