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Two principal modes of cell death are now recognized, namely necrosis and apoptosis. Both may follow cell injury but the nature of the two types of death are quite different.
Mode # 1. Cell Necrosis:
The changes in cells undergoing necrosis are essentially a continuation of those described above, in cell injury. However, they are, of course, more profound and are irreversible. It has thus been proposed that there is a ‘point of no return’, beyond which the process of cell injury cannot be revoked.
While moderate mitochondrial swelling, the formation of cell surface ‘blebs’ and ribosomal disaggregation appear to be reversible, there are certain changes which will inevitably lead to cell necrosis. These include extreme ‘blebbing’ at the cell surface, together with much greater dilation of the mitochondria (‘high amplitude swelling’) and the formation of electron dense areas in the mitochondrial matrix.
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Eventually, there is cell membrane disruption, dissolution of organelles, including the nucleus, and lysosomal degeneration with activation of an inflammatory response. The latter is probably induced by complement-activation by fractions from mitochondria and by leukotrienes formed by lipid peroxidation.
Mode # 2. Apoptosis:
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Apoptosis or ‘programmed cell death’ has been describe for over 20 years but only comparatively recently has its fundamental importance been recognized. It is known to be central to many developmental processes where cells have to be lost as part of the organization of tissues or organs. Examples lie in the loss of tadpole tails, the loss of inter-digital webs, the control of B lymphocyte proliferation and the removal of excess cells in nervous system maturation.
In pathological terms, apoptosis is fundamental to the process of atrophy, where cells are lost from mature organs or tissues as a result of endocrine or physical means. For example, hormones may suppress target glands by this route and surgical ligation of the draining ducts of, for example, the pancreas can lead to cell loss by apoptosis.
One great ‘advantages’ of apoptosis versus necrosis is that there is no induction of acute inflammation and thus further tissue damage is not incurred. This is of particular importance in the removal of neutrophils, whose lysosomal enzymes could continue to cause tissue disruption if left unchecked.
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Apoptosis also appears to be a major mechanism by which malignant cells are removed and imbalances between apoptotic and mitotic rates could lead to tumour progression or regression. The observation that some chemotherapeutic drugs used for treating cancer can act by inducing apoptosis may be of great therapeutic importance and estimates of apoptotic rates may be of value in assessing the efficacy of therapeutic regimes.
The morphological changes accompanying apoptosis are quite different to those seen in necrosis. First, there is loss of cell-cell adhesion and in vitro the affected cells ‘float’ above their normal counterparts. The cells also become rounded and smaller, sometimes with lobuation, although some organelles, including mitochondria, remain intact.
This shrinkage results from loss of Na+ and water. The nuclear chromatin condenses to form ‘half-moon’-shaped structures just within the nuclear membrane and just nucleoli appear disorganized. The nucleus may also break up into multiple portions.
There is an increase in transglutaminase activity in the cell, leading to insolubilization of proteins, which form a ‘shell’ round the inner surface of the cell membrane. There is also a rise in Ca2+, Mg2+ -dependent endonuclease, which is responsible for the chromatin condensation and the nuclear fragmentation.
Finally, the apoptotic cells are rapidly recognized by macrophages, which phagocytose them, with the formation of apoptotic bodies within the cytoplasm of the consuming cells, where they may be seen for up to about 9 hours following phagocytosis. The binding of macrophages to apoptotic cells appears to be mediated by the macrophage vitronectin receptor, which is also a cell adhesion molecule. Binding is also facilitated via a lectin-type receptor on the macrophage surface.
Apoptosis is a genetically regulated phenomenon and has been studied extensively in the nematode worm Caenorhabditis elegans, where mutations of certain gene loci are related to programmed cell death. Thus, mutations of the loci ced-3 and ced-4 lead to the blockage of the apoptosis known to be necessary during development of this organism.
A further gene, ced-9, appears to have some control over the process and it appears that its gene product may either enable or disenable the apoptotic system. Furthermore, ced-9 has been shown to possess homology with the human oncogene bcl-2. This gene is one of several known to regulate apoptosis in human cells.
Techniques for the Detection of Apoptosis:
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The simplest method for the detection of apoptotic cells is observation of the characteristic bodies in tissue sections at either light or electron microscope level. In vitro, the apoptotic cells can be visualized by means of phase or interference contrast microscopy, where they can be seen to be rounded and lying above the cell monolayer.
However, the ‘gold standard’ lies in the demonstration of 180-200 base oligonucleotide fragments resulting from nuclear damage. These fragment scan be separated by gel electrophoresis and viewed under ultraviolet (UV) light, where they produce a characteristic ‘ladder’ effect. This method is applied to cell extracts and has the disadvantage that it is not quantitative.
Other approaches include the use of cell suspensions stained with fluorescent DNA-binding dyes which are then analysed in a DNA flow cytometer. A novel approach is that of DNA end-labelling, in which nucleotides, tagged with a suitable marker, are bound to the broken ends of DNA fragments in apoptotic cells. This method has the advantage that it enables the enumeration of affected cells.