PubMed2000
In the present study, VP16, an inhibitor of topoisomerase II, and NaN3, a chemical toxic substance, were used to induce apoptosis and necrosis of HL-60 cells, respectively. Cells were examined by transmission electron microscopy and by fluorescence microscopy after staining with Hoechst 33258 at 0, 2, 4, 8, 16 and 24 h of culture. Alterations of fluorescent intensity produced by FDA, Rh 123 and PI were measured by flow cytometry, which reflects sequential changes on plasma membrane permeability and the potential of mitochondrial membrane. The results indicated that the nuclear morphologic alteration of cells treated with VP-16 began at 4 hour of culture and the cells with condensed nucleus culminated at 8 h of culture and then reduced with no drop in cell counts, while the percentage of cells with fragmented nuclei reached its maximum at 24 h of culture accounting for some 80%. The potential of mitochondrial membrane of of apoptotic cells decreased gradually from 8 h of culture and showed obvious decline at 16 h of culture. In necrotic cells, the potential decreased by 50% at 4 h of culture which indicates an earlier and more rapid decline than that in apoptotic cells. Alteration of plasma membrane permeability appeared at 8 h of culture and showed a steady increase over time. It was concluded that plasma membrane permeability and mitochondrial membrane potential could reflect the development and degree of apoptosis, and the combination of these two criteria with nuclear morphology revealed by staining with Hoechst 33258 would be a simple and reliable assay for apoptosis and its progression.