# 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