Radiation Oncology/Radiobiology/Cell Death

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Cell Death


Overview[edit]

  • Definition
    • Physiology: physical death of a cell
    • Radiobiology: loss of ability to proliferate indefinitely (clonogenic death)
  • Types of cell death
    • Apoptosis: highly regulated (programmed) process
    • Autophagy: digestion of parts of cytoplasm to generate basic nutrients and to eliminate damaged proteins and organelles
    • Necrosis: death due to extremely unfavorable conditions
    • Senscence: permanent loss of ability to divide
    • Mitotic catastrophe: death following aberrant mitosis
  • Timing after RT
    • Pre-mitotic:
      • Typically within several hours after RT
      • Usually limited to thymocytes, lymphocytes, spermatogonia, and other rapidly proliferating cells
      • Typically via apoptosis
    • Post-mitotic:
      • Cells may progress through one, two or more cell cycles
      • Death may be via several mechanisms, including necrosis and apoptosis
  • After RT, most cells die by mitotic death
  • RT dose
    • Mean lethal dose for loss of proliferative ability <2 Gy
    • Dose to destroy cell function in non-proliferating tissues >100 Gy

Apoptosis[edit]

  • Active cell death, which requires energy, RNA and protein synthesis
  • Fast phagocytosis of well-circumscribed cellular fragments
  • No inflammation, no tissue damage
  • Occurs within 4-6 hours (secondary apoptosis may be seen later at 24-96 hours, in cells undergoing mitotic catastrophe)
  • Function
    • Development of multicellular structures (e.g. sculpting of tissues, regulating neuronal development)
    • Immune response and lymphocyte development
    • Cancer
  • Detection
    • Microscopy: Small darkly stained nuclei, free 3' DNA ends by TUNEL assay
    • Gel electrophoresis: DNA ladder 180 bp intervals
    • Flow cytometry: DNA fragmentation with propidium iodide
    • Cytoplasm: Cytochrome c staining
    • Caspase activity: PARP staining
  • Triggers
    • Extracellular signals: Fas, p75, TNF
    • Toxic stimuli: viruses, chemicals
    • DNA damage: p53
    • Plasma membrane damage: sphingomyelinase
  • Three phases
    • Induction:
      • External pathway from receptors on cell membrane
      • Internal pathway via mitochondria
      • Sphingomyelinase pathway from plasma membrane damage
    • Execution: Caspases, which exist as dimers and are proteolytically activated
    • Degradation: Membrane, cytoplasm, and DNA destruction
  • Extrinsic pathway (Overview)
    • Not induced by RT
    • Fas ligand attaches to FAS receptor ("Death receptor"), which is internalized. Similarly, TNF-related apoptosis-inducing ligand (TRAIL) attaches to death receptors DR4 and DR5
    • Adaptor molecule FADD attaches to the internalized FAS/DR4 to form death inducing signalling complex (DISC)
    • Within DISC, inactive pro-caspase 8 is converted to active caspase 8 (FLICE)
    • Activated caspase 8 then activates caspase 3 through two separate pathways
      • Direct pathway, when concentration of caspase 8 is high: caspase 8 cleaves pro-caspase 3 directly, and activates it
      • Indirect pathway, when concentration of caspase 8 is low: caspase 8 cleaves Bcl-2 interacting protein (Bid), which results in release of cytochrome c from mitochondria.
      • This joins the Intrinsic pathway below
  • Intrinsic pathway
    • Induced by RT after DNA damage, and p53 activation
    • p53 in turn activates the pro-apoptotic members of Bcl-2 protein family (Bax, Bak, Bim, Puma), which are in balance with anti-apoptic members of this family (Bcl-2, Bcl-xL). Together they govern the permeability of mitochondrial membrane, especially to calcium
    • In response to elevated calcium, cytochrome c is released from mitochondria into cytoplasm, and complexes with Apaf-1 to produce the Apoptosome complex (hectamer)
    • Apoptosome complex recruits and activates caspase 9 from procaspase 9
    • Activated caspase 9 within the apoptosome complex activates caspase 3
    • Caspase 3 activates multiple factors:
      • DNA Fragmentation Factor (DFF) causes DNA cleavage
      • Caspase 7 activates PARP (PARP activity can be used as an indicator of apoptosis)
      • Caspase 6 targets nuclear lamins
      • Cytoskeletal proteins
    • Apopototic cell is ultimately removed by phagocytosis, leaving no trace
  • Sphyngomyelinase pathway
    • Radiation damages plasma membrane
    • Can be triggered by IR in absence of DNA damage
    • Acid sphyngomyelinase becomes activated, and hydrolyzes sphingomyelin to ceramide
    • Ceramide activates mitochondrial apoptotic system (see Intrinsic Pathway), probably by interacting with Bax
    • This pathway is particularly expressed in endothelial cells, and may contribute to radiation-induced vascular injury
  • Caspase types
    • Initiator: caspase 8 (extrinsic), caspase 9 (intrinsic), caspase 10 (extrinsic)
    • Executor: caspase 3, caspase 6, caspase 7
  • Promoters
    • Smac (Diablo) inhibits IAPs
  • Inhibitors
    • Bcl-2 counteracts effects of Bid (extrinsic) or Bax/Bak/Bim/Puma (intrinsic)
    • IAPs (inhibitors of apoptosis) block activation of caspase 3 by Apoptosome complex
    • NFkB pathway activates transcription of IAPs and Bcl-2

Necrosis[edit]

  • Passive cell death
  • Triggered by non-physiological circumstances that disrupt normal cellular homeostasis (e.g. hypoxia, poisoning, etc.)
  • Caused by membrane dissolution
  • Cellular material, including degradative enzymes, released into surrounding tissue, leading to inflammation and tissue damage

Autophagy[edit]

  • Programmed cell death
  • Response to nutrient deprivation, hypoxia, crowding, senescene, genotoxic stresses, or simply mechanism to eliminate damaged organelles
  • Organelles and other cell components, including portions of cell membrane and cytoplasm, are sequestered in autophagosomes. These fuse with lysosomes, causing degradation of contents (self-digestion = autophagy)
  • Distinct morphology from apoptosis or necrosis
  • Increased endocytosis, vacuolation, membrane blebbing, and nuclear condensation
  • No caspase activation

Senescence[edit]

  • Depends on p53/p16 driven G1 block
  • Thought to be driven by telomore shortening