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