Etoposide-induced apoptosis in lymphoblastoid leukaemic MOLT-4 cells: evidence that chromatin condensation, loss of phosphatidylserine asymmetry and apoptotic body formation can occur independently.

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Apoptosis is characterised by a series of typical morphological features, such as nuclear and cellular convolution, chromatin condensation and the final disintegration of the cell into membrane-bound apoptotic bodies, which are phagocytosed, by neighbouring cells. Relocation of phosphatidylserine residues from the inner leaflet of the cellular membrane to being exposed on the cell surface is a necessary event for the phagocytic elimination of apoptotic cell debris. Using the MOLT-4 lymphoblastoid leukaemic cell line we investigated whether the formation of apoptotic bodies and loss of phosphatidylserine asymmetry were causally related. We have previously demonstrated that classical apoptotic morphology, including production of apoptotic bodies, was only possible in etoposide-treated MOLT-4 cells when administered in the presence of non-cytotoxic doses (200 microM) of aurin tricarboxylic acid (ATA). Electron microscopic analysis, followed by the quantitation of the ultrastructural morphological features of apoptotic MOLT-4 cells, demonstrated that the etoposide and ATA co-treatment, which caused the cellular fragmentation into apoptotic bodies, was closely associated with extensive chromatin condensation in individual cells. In this model however, the addition of ATA to frank cytotoxic doses of etoposide (50 microM), which we confirmed lead to formation of apoptotic bodies, caused no further increase in externalisation of phosphatidylserine moieties as determined by staining with fluorescence labelled annexin V. Consequently, in MOLT-4 cells undergoing etoposide-induced apoptosis, the mole-cular mechanisms leading to loss of phosphatidylserine asymmetry and the formation of apoptotic bodies are not causally related.