Cell Toxicity/ Apoptosis/ Inhibition of Proliferation
Cell death, overt death or inhibition of growth, occurs by several mechanisms, including necrosis and programmed cell death, most notably apoptosis. Necrosis is generally caused by hostile influences outside the cell designed to kill the cell and is usually initiated by toxic attack from the outside. Apoptosis in normal organism life is caused by environmental influences desiring the removal of the cell in the interest of the overall organism. These influences lead to several mechanisms of eventual death which are preprogrammed and thus the toxic mechanisms are initiated intra-cellularly.
Necrosis can be initiated by the host organism, such as Natural Killer (NK) lysis or immune T lymphocytes removing a virus-infected cell, by a toxic agent such as a chemical, Xrays, heat, etc., or by pathogen, or by a therapeutic exercise such as Antibody Dependent Cellular Cytotoxity (ADCC), or cancer chemotoxic drug. The initial direct action can be at the level of the cell membrane or an effect on metabolism, e.g. protein, RNA or DNA synthesis.
Necrosis is characterized by loss of plasma membrane integrity, cellular and organellar swelling, and marked inflammation, leading to disruption of the cellular and nuclear membranes. ATP levels are dramatically reduced in necrotic cells, both because of severe mitochondrial damage that cripples ATP generation as well as unrestrained energy expenditures.
Apoptosis is characterized by cell shrinkage, DNA cell disintegration, fragmentation into membrane-enclosed apoptotic bodies, and phagocytosis of these corpses by macrophages, or occasionally, neighboring cells. When this clean-up operation is efficient, inflammation is avoided. ATP levels in apoptotic cells are reasonably well maintained both because of continued production and decreased expenditures. The net result of apoptosis is the stealth deletion of individual cells within a tissue.
Methods of Detection
Overt cell death is assessed microscopically by uptake of trypan blue dye or a DNA stain that is excluded by live cells. High throughput measurement of cell death is performed by release of a label from cells pre labeled with a radiotracer, typically 51Cr or a fluorescent or color marker, or a homogeneous assay (no wash steps) that measures metabolic activity (intracellular ATP content or redox state).
Apoptosis leads to cell death after some days’ delay. Specific measurement of apoptosis includes DNA fragmentation, activation of capase enzymes, and exteriorization of phosphatidylserine groups detected by Annexin V.
An another example of a homogeneous assay is the measurement of intracellular ATP using CellTiter Glo reagent (see below). This is a very sensitive assay for small numbers of cells or small differences in growth and inhibited cell cultures.