KINETICS OF APOPTOSIS AND SECONDARY NECROSIS IN CULTURED RAT THYMOCYTES AND S.49 MOUSE LYMPHOMA AND CEM HUMAN LEUKEMIA-CELLS

Cell culture systems are widely used to study metabolic changes during apoptosis. In cell culture, unlike in vivo, apoptotic cells are not phagocytosed and eventually lyse (secondary necrosis). This is of practical importance because metabolic changes seen in cultures may be due to the transition from apoptosis to necrosis, rather than to the induction of apoptosis itself. In the present study, we followed the kinetics of the occurrence of several indicators of cell death in rat thymocytes and mouse lymphoma (S.49), and human leukemia (CEM) cell cultures after dexamethasone treatment (10(-6) M). The presence of apoptosis and secondary necrosis was demonstrated by electron microscopy. Nuclear condensation and fragmentation, which are considered to reflect early stages of apoptosis, were visualized with Hoechst fluorescent dye H 33258 for quantitative determination by light microscopy. In S.49 and CEM cultures their incidence increased after glucocorticoid treatment, but remained at relatively low levels not exceeding 6-9% until 36 h (S.49) or 3-4% until 92 h (CEM). The trypan blue positive cells, however, increased steadily to about 60%. Furthermore, flow cytometry (single parameter DNA analysis after propidium iodide staining) revealed the occurrence of cells with reduced DNA fluorescence. Morphological and biochemical (internucleosomal DNA cleavage) analysis of FAGS-sorted cells showed that early after dexamethasone the majority of them were apoptotic. In S.49 and CEM cell cultures no clear-cut time lag between increase in cells with reduced DNA fluorescence, chromatin condensation/fragmentation, and the uptake of trypan blue could be detected. Rat thymocytes, however, exhibited a time lag between the increase in cell number with reduced DNA fluorescence and trypan blue index, but only until 8 h after treatment. Thereafter, again no clear-cut time lag between these indicators of cell death could be observed. This suggests that, at least in thymocyte cultures, secondary necrosis ensues apoptosis and both can be discriminated for a short period of time. Within this period of time we could detect increased levels of transglutaminase mRNA levels. On the other hand, clusterin mRNA levels did not increase after dexamethasone treatment. Taken together, these findings suggest that rat thymocytes, S.49, and CEM cells enter apoptosis asynchronously during the entire period of investigation and then rapidly progress into secondary necrosis.