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
 * Senescence: 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 ([Broken Link: 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, senescence, 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 telomere shortening
 * Cells do not divide but remain metabolically active

Mitotic Catastrophe

 * A type of cell death that occurs during mitosis
 * Proposed resulting from a combination of deficient cell-cycle checkpoints (in particular the DNA structure checkpoints and the spindle assembly checkpoint) and cellular damage
 * Controlled by numerous molecular players, in particular, cell-cycle-specific kinases (such as the cyclin B1-dependent kinase Cdk1, polo-like kinases and Aurora kinases), cell-cycle checkpoint proteins, survivin, p53, caspases and members of the Bcl-2 family