Etoposide induces DNA damage that signals for various forms of active call death in various eel! types, that frequently differ from each other in morphology, in kinetics and in the molecules involved. Here we show, using a specific caspase inhibitor, that several forms of active death can be discriminated in one model, a lymphoma cell line, exposed to etoposide. IJT58, a human B lymphoma cell line was treated with 10pM etoposide and/or 100 pM Z-VAD-FMK as a general caspase inhibitor, which was given daily. Viability was assessed by trypan blue staining. Cell cycle arid sub-Gl (apoptotic) cell population were measured by flow cytometry after propidium iodide staining. Clonality assay (12 days) was performed in soft agar. HT58 cells exposed to etoposide died by fast apoptosis (at 4 h) dependent on caspase activity but independent of new protein synthesis. More prolonged (3 days) incubation with etoposide resulted in apoptosis, but with a slower rate, that was also sensitive to caspase inhibitor, Z-VAD-FMK. HT58 cells are Fas receptor (FasR) negative, but etoposide treatment induced FasR expression on the first day indicating the possible involvement of FasR mediated signals in cell death. In a third phase (after the 3rd day) a portion of the etoposide treated, Z-VAD-FMK-rescued lymphoma cells died in spite of the presence of fresh caspase inhibitor. The survived cells were arrested in S phase, that may protect cells from engagement to mitotic death in the G2 phase. However, the cells protected by caspase inhibitor against etoposide lost their clonal potential (</% of control), similar to those 10% of cells that survived the etoposide only treatment. Our results support a three phase model in etoposkle-iuduced cell death: 1. protein synthesis independent fast apoptosis of S-G2 celts with pivotal role of caspases at 4 h; 2. slower death-phase also dependent on caspase activity possibly requiring death receptor expression (1-3 days); . caspase-independent death-phase (after 3 days) with unknown mechanism.
|Number of pages||6|
|Publication status||Published - Dec 1 1999|
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