Degradation of apoptotic cells and fragments in HL-60 suspension cultures after induction of apoptosis by camptothecin and ethanol.
Early indicators of apoptosis in mammalian cells are membrane potential breakdown (loss) in mitochondria (MPLM), chromatin condensation, DNA degradation, and phosphatidylserine exposure (PSE) on the outside plasma membrane. One aim of the present study was to determine the kinetics of these characteristics. These changes were measured by flow cytometry using the following methods: membrane potential of mitochondria was analysed using Mito Tracker Green and Red, PSE was analysed using annexin-V-FITC staining simultaneously with propidium iodide (PI) to detect membrane permeability; chromatin condensation was measured using the acid denaturation Acridine Orange (AO) method; DNA degradation was studied by the sub G1 method and the terminal transferase dUTP nick end-labelling (TUNEL) assay (labelling of strand breaks). HL-60 cells were induced to apoptosis by 3% ethanol and 1.5 microM camptothecin (CAM) and the kinetics of the apoptotic cells were measured. The same kinetics were found for chromatin condensation and DNA degradation indicating that these changes appeared at approximately the same time after induction. The MPLM and PSE kinetics showed a considerably later increase indicating that MPLM occurred downstream of DNA degradation and that plasma membrane changes occurred downstream of MPLM. The main aim of the study was to follow the fate of apoptotic cells after the appearance of the initial characteristics. The lifetime of apoptotic cells was studied by chase experiments. The inducing drug was removed after 4 h treatment and the disappearance of apoptoses recorded. An exponential decay was measured with a half life (T(1/2)) of 17.8 h. As a corollary from these experiments, camptothecin was found to induce apoptosis also in G1 and G2 phase cells, however, it took much longer to occur than in S phase cells. Using labelling of the plasma membrane with a fluorescent cell membrane linker, it was possible to show that the majority of apoptotic bodies as well as condensed apoptotic cells contain DNA and membrane. The degradation of these apoptotic bodies follows similar kinetics as those of the condensed apoptotic cells. The membrane remained considerably stable, there was no further loss in the next 7 days, after the first day when the apoptotic characteristics develop. It is concluded that the apoptosis programme is completed within a day and no further steps follow.
Baisch
H
H
Institute of Biophysics and Radiobiology, University of Hamburg, Germany. baisch@uke.uni-hamburg.de
Bollmann
H
H
Bornkessel
S
S
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002166:Camptothecin / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D018922:HL-60 Cells; D006801:Humans; D020287:In Situ Nick-End Labeling; D007700:Kinetics
D005456:Fluorescent Dyes; D000431:Ethanol; D002166:Camptothecin