Radiation Oncology/Radiobiology/Cell Death

Front Page: Radiation Oncology | RTOG Trials | Randomized Trials

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