“Cell death” in radiobiology means that a cell is rendered incapable of regenerating the tumour mass. This can be due to destruction of the cell through apoptosis, autophagy, or necrosis. It may also occur if the cell is made senescent or if it undergoes mitotic catastrophe.
Apoptosis
Apoptosis is the regulated form of cell death that occurs in response to numerous extra- or intracellular signals. These can include hypoxia, excessive oncogene activation, or cytokines from neighbouring cells. It can also be induced by natural killer cells through introduction of granzyme-B into the cell.
The Intrinsic Pathway
The intrinsic pathway occurs in response to internal stimuli. Apoptosis is held in check by members of the BCL-2 family of genes. BAX and BAK are members of the pro-apoptotic group whose expression is increased due to internal stimuli like DNA damage or hypoxia. They function by increasing the permeability of the mitochondrial membrane and allowing the efflux of cytochrome C and caspase 9. BCL2 and BCL-XL are anti-apoptotic proteins which prevent BAX and BAK from interacting with the mitochondrial membrane. A group of BH3-only BCL-2 proteins help to regulate the balance of the pro- and anti-apoptotic proteins.
The Extrinsic Pathway
The extrinsic pathway involves stimulation of cell surface death receptors, which trigger the release and activation of caspase 8. Caspase 8 cleaves caspase 3, the most well understood executioner caspase, that cleaves numerous proteins and leads to apoptosis.
The Granzyme Pathway
The other mechanism occurs through influx of granzyme-B which is injected into the cell by natural killer cells and a specific cell membrane channel. Granzyme-B activates the caspases and leads to apoptosis.
Ceramide
This less well understood mechanism relies of ceramide, a lipid molecule that exists in the cell membrane. Due to a number of stimuli (including radiation and cytokine signalling), ceramide may be released from the membrane and cause pro-apoptotic effects. The exact mechanisms are not fully understood, but may involve activation of the intrinsic pathway, or migration of ceramide to the mitochondrial membrane where it forms pores and causes an efflux of caspase 8.
Appearance of Apoptosis
Apoptosis is visually distinct from the other forms of cell death. The initial step is the separation of the cell from its neighbours (to prevent cross contamination of caspases). The nucleus and cytoplasm condense. The cell membrane forms characteristic blebs, and the nucleus fragments into pieces. The blebs of cytoplasm (apoptotic bodies) may or may not contain nuclear fragments. These remains are digested by macrophages to prevent inadvertent exposure of cytoplasmic and nuclear proteins to antigen presenting cells.
There is a lack of an acute inflammatory response following this mechanism of cell death, due to 'hiding' of the intracellular components from the immune system.
If the DNA of apoptotic cells is run in a gel electrophoresis, a characteristic laddering pattern is seen. This is due to the fragmentation of DNA into specific lengths.
Variations in Apoptosis
The level of activity required for the intrinsic pathway to lead to apoptosis is regulated differently in alternate cell types. Lymphocytes are often very sensitive to TP53 activation from ionising radiation, leading to their rapid death following exposure. Fibroblasts do not undergo apoptosis despite TP53 and BAX activation, possibly due to increased activity of anti-apoptotic proteins. Tumour cells often deactivate TP53 or alternately increased expression of BCL-2, thus preventing apoptosis from occurring.
Autophagy
Autophagy is also a form of programmed cell death, but is also used by the cell when it is deprived of nutrients or stimulated by some growth hormones. ATG proteins are expressed in response to mTOR activation (which occurs in low nutrient states). These molecules form a double membrane bound structure which envelops cytoplasmic contents. The structure then fuses with a lysosome, leading to degredation of the contents and release of energy.
Autophagy does not require caspases and is morphologically distinct from apoptosis. It seems to function as another method of tumour suppression as mutations in the component genes are seen in some cancers. It is seen after treatment with anti-cancer agents, including radiotherapy, particularly when the apoptosis pathway is damaged.
Necrosis
Necrosis is usually considered an uncontrolled form of cell death due to loss of nutrients or other critical problems in the cellular environment. Recent studies have shown that it may another form of regulated death in response to critical injuries, as TP53 may be increased in expression prior to necrosis occurring. Degredation of lysosome structure leads to release of enzymes that attack and destroy the cell proteins.
Following radiation, necrosis is seen less frequently but does occur in some cell lines.
Senescence
Cellular senescence refers to the loss of ability to divide that is seen in normal cells after numerous divisions. It is associated with the shortening of telomeres.
Premature senescence is activated in response to a number of cellular stressors, including ionising radiation and DNA damage. The mechanism/s are not fully are understood, but TP53, RB1 and CDKI are all thought to be involved. The cell is placed in a permanent G0 state, characterised by increased gene silencing and chromatin changes. These pathways are often mutated in malignancies to prevent senescence from occurring.
Mitotic Catastrophe
If a cell proceeds through mitosis without proper alignment of chromosomes on the metaphase plate, the division of the cell may lead to aneuploidy in both daughter cells. This is known as mitotic catastrophe as the cells die due to loss (or gain) of significant genetic material. This may be due to loss of genes that allow mitosis to occur or due to inability of the cell to pass on genetic material once the catastrophe has occurred. Other forms of cell death may occur in response to a mitotic catastrophe.
Loss of cell checkpoints (G2 and M phases) lead to increased risk of mitotic catastrophe, and are more common in tumours. Mitotic catastrophe is thought to be perhaps the most important form of cell death following ionising radiation. Cell death is not immediate; cells may smply become senescent (unable to divide), or the may divide several more times for permament cell death takes place.
