Sizing highly fragmented DNA in individual apoptotic cells using the comet assay and a DNA crosslinking agent

TK6 human B lymphoblast cells exposed to ionizing radiation undergo apoptosis in a time and dose-dependent manner. The resulting highly fragmented DNA is easily detected using the comet assay, a sensitive microscopic gel electrophoresis method capable of measuring DNA strand breaks in individual cells. The degree of DNA fragmentation may be indicative of different stages in the fragmentation process, responses to different agents, and/or cell type-dependent differences. In an effort to determine the number of breaks present in each apoptotic cell, we first applied a DNA-crosslinking agent, mechlorethamine, to TK6 cells containing a known number of DNA double-strand breaks produced by X rays. As the concentration of mechlorethamine increased, crosslinked DNA was less able to migrate during gel electrophoresis. Exposure of TK6 cells to 5 microM mechlorethamine prior to irradiation with 20 Gy was sufficient to "hide" the presence of these breaks by preventing DNA from migrating during electrophoresis. However, in apoptotic TK6 cells, it was necessary to apply a dose of mechlorethamine several times higher in order to produce a similar degree of inhibition of DNA migration. Calibrations using either the alkaline or neutral comet assays indicate that the average DNA fragment size in apoptotic TK6 cells is about 50 kb. Even in cells containing only 10-20% of the original amount of DNA, the remaining fragments still averaged about 50 kb, indicating that fragmentation to much smaller sizes occurs in some parts of the genome before others. When Chinese hamster V79 cells were exposed to hyperthermia (45 degrees C for 20 min), necrosis was induced over a period of several days. The size of DNA fragments in these cells was considerably larger (200-400 kb) and heterogeneity in appearance of comets was larger than observed for TK6 cells. This crosslinking method may be useful in discriminating cells dying by apoptosis from cells damaged or dying by other mechanisms.