Etoposide at different concentrations may open different apoptotic pathways in thymocytes

Apoptosis of thymocytes has been investigated by morphological, biochemical and cytometric techniques, both in non-perturbed conditions and after exposure to the topoisomerase-II inhibitor etoposide in vitro. Apoptotic thymocytes were already present in untreated samples, and their frequency increased with increasing drug concentrations. Using flow cytometry, we demonstrated that the frequency of apoptotic thymocytes in S and G2 phase of the cell cycle was higher in etoposide-treated samples, consistent with the phase-preferential action proposed for this topoisomerase-II inhibitor. At electron microscopy, the sequence of nuclear events was apparently independent from the apoptogenic trigger, so that similar features of chromatin margination and nuclear fragmentation were observed in thymocytes undergoing apoptosis both spontaneously and after etoposide treatment. The morphology and function of plasma membrane were still preserved in apoptotic cells. At low (0.1 to 10 microM) etoposide concentrations apoptotic cells had cytoplasm patterns similar to naturally occurring apoptotic thymocytes, whereas at high (50 to 100 microM) concentrations extensive organelle damage took place. Damaged cytoplasm was sometimes polarized into discrete portions, which were eventually extruded within apoptotic bodies. This evidence suggests that, in the same cell type, different apoptotic pathways are opened by this agent, depending on the concentration used.