APOPTOSIS
APOPTOSIS
- Programmed cell death.
- Self-initiated & energy-dependent process.
- Eliminates irreparable & damaged cells.
CAUSES OF APOPTOSIS
PHYSIOLOGICAL
- Natural phenomena
- Embryogenesis
- Involution of hormone-dependent tissues (examples are endometrium & prostate )
- Ageing.
- Cell death in proliferating cell population (eg: interstitial epithelium).
- Deletion of harmful self-reacting lymphocytes (mechanism to prevent autoimmunity).
PATHOLOGICAL
- DNA damage due to radiation
- Cytotoxic anticancer drugs
- Extremes of temperature
- Accumalation of misfolded protein
- Cell injury in certain infections as in HIV
- Pathological atrophy of glands after ductal obstruction.
- Tumor cell death.
MORPHOLOGICAL FEATURES:
- Cell shrinkage –
- Earliest change
- Due to damage to cytoskeletal proteins.
- Chromatin condensation (pyknosis)/nuclear compaction
- Most characteristic feature.
- Convolution of cell membrane
- Leading to formation of cytoplasmic blebs (although cell membrane remains intact).
- Formation of apoptotic bodies:
- Membrane-bound spherical bodies containing compacted organelles with/without nuclear fragments.
- Examples- Civatte bodies, kamino bodies, councilman bodies (as in viral hepatitis).
- Does not elicit any inflammatory response.
- Due to intact cell membrane.
- Ultimately phagocytosis of these bodies.
MECHANISM OF APOPTOSIS
- Caspase acivates endonuclease (neuronal apoptosis lacks caspases, thus activation of AIF)
- Endonuclease damages DNA
- Chromatin clumping
INITIATION PHASE
- Consists of two pathways –
Intrinsic/Mitochondrial Pathway
- Major mechanism.
- Cellular stress/injury activates stress sensors (BH3 proteins).
- Acts by 2 ways:
- Activation of PRO-APOPTOTIC PROTEINS (BAX,BAK,p53).
- Inactivation of ANTI-APOPTOTIC PROTEINS (Bcl-2, MCL-1) on outer membrane of mitochondria.
- In turn results in increased mitochondrial permeability.
- “Cytochrome c” leaks out to cytoplasm.
- Leakage of “SMAC /DIABLO” into cytoplasm.
- It binds to APOPTOSIS activating factor 1.
- Inhibits ANTI -APOPTOTIC IAP – Physiological inhibitor of apoptosis.
- Forms apoptosome complex.
- Triggers CASPASE 9 activation
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
- eg: Type1 TNF receptors & FAS (= CD95)
- FAS (receptor) + FAS-L (ligand)
- Trimerisation occurs and activation of adaptor protein
- Activation of CASPASE 8 &10 (in humans).
- CD 95 –Marker of extrinsic pathway of Apoptosis.
EXECUTION PHASE:
- Initially caspases will activate,
- Executioner caspases: caspase-3 and caspase-7
- Disrupt cytoskeleton & leads to cell fragmentation.
- Finally phagocytosed by macrophages
DYSREGULATED APOPTOSIS
- Defective apoptosis and increased cell survival – autoimmune diseases
- Increased apoptosis and excessive cell death
- Neurodegenerative diseases
- Ischemic injury (MI, stroke)
- Viral infections
IDENTIFICATION OF APOPTOSIS
- Staining of chromatin condensation.
- Flow cytometry –Most commonly used method for detecting apoptosis.
- To visualize rapid cell shrinkage.
- DNA changes detected by in situ techniques or by gel electrophoresis.
Agarose gel electrophoresis:
- Endonucleases induced inter-nucleated damage – Appearing as “Step-ladder pattern”.
Annexin V Assay:
- Classical technique for detecting apoptosis.
- Annexin V –
- Calcium-dependent phospholipid-binding protein.
- Has high affinity for phophatidylserine (PS), plasma membrane phospholipid.
Earliest features:
- Translocation of PS from inner to outer leaflet of plasma membrane → exposing PS to external environment.
- Annexin V binds to this exposed PS.
- Thus, identifies cells at earlier stage of apoptosis (When compared to assays based on DNA fragmentation.
Exam Important
- 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 Cytochrome c from 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 & immune cytolysis.
- The earliest change seen in apoptosis is Cell shrinkage.
- Execution caspases of apoptosis are Caspase 3 & 7.
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