ETOPOSIDE AT DIFFERENT CONCENTRATIONS MAY OPEN DIFFERENT APOPTOTIC PATHWAYS IN THYMOCYTES

Apoptosis of thymocytes has been investigated by morphological, bioche mical and cytometric techniques, both in non-perturbed conditions and after exposure to the topoisomerase-II inhibitor etoposide in vitro. A poptotic thymocytes were already present in untreated samples, and the ir frequency increased with increasing drug concentrations. Using flow cytometry, we demonstrated that the frequency of apoptotic thymocytes in S and G(2) phase of the cell cycle was higher in etoposide-treated samples, consistent with the phase-preferential action proposed for t his topoisomerase-II inhibitor. At electron microscopy, the sequence o f nuclear events was apparently independent from the apoptogenic trigg er, so that similar features of chromatin margination and nuclear frag mentation were observed in thymocytes undergoing apoptosis both sponta neously and after etoposide treatment. The morphology and function of plasma membrane were still preserved in apoptotic cells. At low (0.1 t o 10 mu M) etoposide concentrations apoptotic cells had cytoplasm patt erns similar to naturally occurring apoptotic thymocytes, whereas at h igh (50 to 100 mu M) concentrations extensive organelle damage took pl ace. 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 ar e opened by this agent, depending on the concentration used.