The relevance of apoptosis for cellular homeostasis and tumorigenesis: MORPHOLOGY AND DETECTION Part 2
Identification and quantification of apoptosis: The number of techniques available for identifying apoptosis are summarized in Table 1. Because apoptosis is defined by a series of distinct changes in cellular morphology, light and electron microscopy provide the best evidence for detecting and quantifying apoptosis in intestinal epithelium. Many contemporary studies on intestinal apoptosis supplement traditional microscopy with in situ techniques for the detection of broken DNA strands. In the strictest sense, ‘programmed cell death’ may be applied to circumstances where death is initiated by a genetic program that leads to autonomous cell destruction. It is now recognized that a cell may undergo ‘programmed cell destruction’ without fulfilling some, or all, of the morphological criteria of apoptosis. When considering apoptosis in the intestinal epithelium, this is not just a matter of semantic debate. For example, epithelial cells are shed from the tips of the intestinal villi. Because the dimensions of the villi tips are remarkably constant, it seems reasonable to assume that the shed cells are undergoing a form of programmed cell death. However, cells complying with the strict morphological definition of apoptosis are rarely seen at the tip of the intestinal villus, raising the question of whether other mechanisms account for cell shedding.
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TABLE 1
Techniques for the detection of apoptosis
| Traditional microscopy |
| Light microscopy |
| Electron microscopy |
| Acridine orange fluorescence |
| Biochemical approaches |
| Agarose gel electrophoresis |
| Pulsed field electrophoresis |
| Flow cytometry |
| Unfixed cells |
| Fixed cells |
| In situ detection of broken DNA strands |
| 5-triphosphate nick end-labelling (TUNEL) |
| In situ end-labelling (ISEL) |
| Recently established techniques |
| Annexin V immunohistochemical expression |
| Clusterin immunohistochemical expression |
| In situ hybridization using digoxigenin-labelled poly (A) oligonucleotide probes |
References appear in parentheses
When quantifying apoptosis by morphological features, other caveats to the technique should be borne in mind. The rapid nature of apoptosis means that in any static analysis, a very small number of apoptotic cells observed at a given instant might, in fact, reflect a very considerable contribution to cell turnover. There are recognized variations in the speed of apoptosis among cell types and in relation to different insults. For example, the halflife of apoptotic fragments following treatment with hydroxyurea is approximately 3.5 h, while following radiation it is 15 h (data from our laboratory). A caveat to these remarks is that such half-life measurements relate to the removal of apoptotic bodies by phagocytic digestion and cell migration combined with the true duration of the cell death process. Hall and Coates have described a counting technique termed the ‘wandering mean method’ in an attempt to overcome some of these limitations.
Category: Gastroenterology
Tags: Apoptosis, bcl-2 family, Gastrointestinal cancer, Intestinal crypt, p53, Stem cell