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 p hagocytosed and eventually lyse (secondary necrosis). This is of pract ical importance because metabolic changes seen in cultures may be due to the transition from apoptosis to necrosis, rather than to the induc tion of apoptosis itself. In the present study, we followed the kineti cs of the occurrence of several indicators of cell death in rat thymoc ytes and mouse lymphoma (S.49), and human leukemia (CEM) cell cultures after dexamethasone treatment (10(-6) M). The presence of apoptosis a nd 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 3 3258 for quantitative determination by light microscopy. In S.49 and C EM 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 b iochemical (internucleosomal DNA cleavage) analysis of FAGS-sorted cel ls showed that early after dexamethasone the majority of them were apo ptotic. In S.49 and CEM cell cultures no clear-cut time lag between in crease in cells with reduced DNA fluorescence, chromatin condensation/ fragmentation, and the uptake of trypan blue could be detected. Rat th ymocytes, however, exhibited a time lag between the increase in cell n umber with reduced DNA fluorescence and trypan blue index, but only un til 8 h after treatment. Thereafter, again no clear-cut time lag betwe en these indicators of cell death could be observed. This suggests tha t, 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 m RNA 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 asynchronousl y during the entire period of investigation and then rapidly progress into secondary necrosis.