MORPHOLOGICAL ASSAYS OF APOPTOSIS
The original descriptions3 characterized apoptosis as a form of cell death with distinct morphological features, which include shrinkage of the cell; detachment from its neighbors; condensation of the chromatin beginning in the areas adjacent to the inner nuclear membrane; fragmentation of the nucleus into multiple chromatin bodies, each surrounded by remnants of the nuclear envelope; packaging of the cell into multiple plasma membrane-enclosed cellular fragments; and phagocytosis of these fragments by neighboring cells.4-5 Among these changes, chromatin condensation and nuclear fragmentation are the easiest to assess morphologically.
The chromatin condensation reflects, at least to a first approximation, the action of nucleases that are activated during apoptosis.6-7 Nuclear fragmentation requires cleavage of the lamins, major structural proteins of the nuclear envelope, by caspases.8,9 Thus, the morphological changes described above reflect biochemical alterations that occur in apoptotic cells. These morphological changes can be assessed by electron microscopy, by conventional light microscopy, or by fluorescence microscopy, after staining of the DNA. Each of these approaches has advantages and disadvantages.Electron Microscopy
The preceding description of apoptotic morphological changes was initially based on electron microscopy of cells after exposure to a variety of toxic stimuli.4 Application of this technique to relatively uniform cell populations such as tissue culture cell lines or lymphoid cells undergoing apoptosis after treatment with proapoptotic stimuli permitted precise description and temporal
FIGURE 3.1 (Continued) (B) Detection by fluorescence microscopy after DNA staining. Jurkat cells were treated for 24 h with diluent (top) or 2.5 μM etoposide (bottom).
After fixation in 3:1 (v:v) methanol:acetic acid, cells were dropped onto coverslips, treated with 1 μg∕ml Hoechst 33258 in 50% glycerol containing 50 mM Tris-Hcl (pH 7.4), and examined by fluorescence microscopy. Compared to nonapoptotic cells (top panel and arrowhead, bottom panel), etoposide-treated cells show chromatin condensation (bright staining) and nuclear fragmentation (arrow). (C) Detection by agarose gel electrophoresis. HL-60 human acute myelomono- cytic leukemia cells were treated for 6 h with diluent (left lane) or 68 μM etoposide (right lane). At the completion of the incubation, total cellular DNA was prepared by lysing cells in SDS containing 1 mg/ml proteinase K, extracting the resulting peptides in phenol/chloroform, and directly applying the DNA from 5 ? 105 cells to nonadjacent lanes of a single 1.2% (w∕v) agarose gel (see Kaufmann, S.H., Cancer Res., 49, 5870, 1989.) Before drug treatment, the DNA runs at the exclusion limit of the gel. After induction of apoptosis, the DNA yields a typical nucleosomal ladder consisting of ~180 bp multiples beginning at 180 bp. (D) Detection of particles containing can also be detected in tissue culture by staining with a DNA binding dye, such as Hoechst 33258 or 4,6-diamidino-2-phenylindole (DAPI), and examining cells under a fluorescent microscope. Hallmark changes of chromatin condensation and nuclear fragmentation are readily visible by this technique (Figure 3.1B). For some dyes, such as Hoechst 33258, permeabilization with a detergent or organic solvent is required for the dye to enter the cell. For others (e.g., DAPI), dye uptake into nonpermeabilized cells is sufficient for the fluorescence of highly condensed nuclei to be readily visible.Like light microscopy, this approach has the advantage that a large number of cells can be examined, potentially making quantitation of the number of apoptotic cells more accurate than electron microscopy. If this approach is used to examine slides prepared from tissue culture cells, however, it is important to assess whether apoptotic cells are uniformly distributed throughout the slide. In addition, when fluorescence microscopy is applied to tissue sections, nuclei of quiescent cells (e.g., small lymphocytes) must be carefully distinguished from apoptotic nuclei.
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