KINETICS OF PLASMA-MEMBRANE AND MITOCHONDRIAL ALTERATIONS IN CELLS UNDERGOING APOPTOSIS

Programmed cell death or apoptosis is characterized by typical morphol ogical alterations. By transmission electron microscopy, apoptotic cel ls are identified by condensation of the chromatin in tight apposition to the nuclear envelope, alteration of the nuclear envelope and fragm entation of the nucleus, whereas integrity of the plasma membrane and organelles is preserved, Conversely cells undergoing necrosis display an early desintegration of cytoplasmic membrane and swelling of mitoch ondria, In this study we assessed by now cytometry the sequential alte rations of forward angle Light scatter, 90 degrees light scatter, and fluorescence associated with fluorescein diacetate, rhodamine 123, and propidium iodide in two human B cell lines undergoing apoptosis induc ed by the topoisomerase II inhibitor VP-16. The kinetics of these modi fications were compared to those of cells undergoing necrosis induced by sodium azide, At the same time intervals, cells were examined by tr ansmission electron microscopy and by UV microscopy after staining wit h Hoechst 33342, We report that sequential changes in Light scatters a nd fluorescein diacetate are similar in cells undergoing apoptosis or necrosis, whereas apoptosis is characterized by a slightly delayed dec rease of mitochondrial activity as assessed by rhodamine 123 staining, Surprisingly a part of cells undergoing apoptosis displayed an early uptake of propidium iodide followed by a condensation and then a fragm entation of their nuclei, It is concluded that uptake of propidium iod ide is a very early market of cell death which does not discriminate b etween necrosis and apoptosis. Along with biochemical criteria, nuclea r morphology revealed by staining with Hoechst 33342 would seem to be of the most simple and most discriminative assay of apoptosis. (C) 199 5 Wiley-Liss, Inc.