Radiation Oncology/Radiobiology/Cell Death

Cell Death

Overview

• 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

• 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

• 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

• 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

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