M. Cejna et al., KINETICS OF APOPTOSIS AND SECONDARY NECROSIS IN CULTURED RAT THYMOCYTES AND S.49 MOUSE LYMPHOMA AND CEM HUMAN LEUKEMIA-CELLS, Biochemistry and cell biology, 72(11-12), 1994, pp. 677-685
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.