Key morphologic changes and DNA strand breaks in human lymphoid cells: Discriminating apoptosis from necrosis

Apoptosis is an important form of physiologic cell death displayed by an enormous variety of tissues under divergent conditions. The recent attention toward apoptosis in virtually all aspects of modern biology indicates that rapid and accurate differentiation between apoptosis and necrotic death is of considerable interest. Apoptosis is distinguishable from necrosis on the basis of several criteria. In this study, we undertook to examine the effects of mild hyperthermia (43°C leading to apoptotic death) and severe hyperthermia (50°C leading to necrotic killing) on associated DNA fragmentation. Using laser scanning and fluorescent microscopic evaluation of DNA “comets” in the single cell gel assay, we compared necrotic and apoptotic DNA damage in a variety of human leukemia and lymphoma cell lines at the level of the individual cell. We show that necrotic cells do display detectable DNA damage. We confirm our preliminary report that comet “tail moment” is sufficient to distinguish between necrotic and apoptotic DNA damage, while comet tail length may confuse the two forms. We report that a recovery period is necessary for expression of increasing apoptotic but not necrotic DNA damage. We show that apoptosis increases with prolonged hyperthermia and confirm that the mode of death changes from apoptosis to necrosis with higher heat loads, producing a greater fraction of cells showing damage. In addition, we show that for necrotic cells, DNA tail moment reflects sensitivity to prolonged exposure without a concomitant change in tail length.