Temporospatial sequence of cellular events associated with etoposide-induced neuronal cell death: Role of antiapoptotic protein Bcl-X-L

Etoposide-induced death comprises such nuclear events as the formation of t opoisomerase II-DNA cleavable complex and cytosolic events including caspas e activation. By first establishing the temporospatial death sequence trigg ered by etoposide in a neuronal cell line, MN9D overexpressing Bcl-X-L (MN9 D/Bcl-X-L) or control vector (MN9D/Neo), we examined whether formation of t his complex is primarily responsible for cell death and at which strategic points and how Bcl-X-L blocks etoposide-induced neuronal death. Etoposide i nduced death that was dependent on caspase, cycloheximide, and calpain in M N9D/Neo cells. Etoposide also induced death in enucleated MN9D/Neo cells, a lthough this was less severe. The level of topoisomerase II-DNA cleavable c omplex reached at a maximum of 2 hr after etoposide treatment was identical in MN9D/Neo and MN9D/Bcl-X-L cells. In MN9D/Neo cells, cytochrome c releas e into the cytosol and caspase activation occurred as early as 2 hr and 3-6 hr after etoposide treatment, respectively. Etoposide-induced DNA ladderin g potentially via caspase appeared as early as 12 hr after drug treatment, followed by nuclear swelling in MN9D/Neo cells (> 18-20 hr). Subsequently, nuclear condensation started by 24-28 hr and became apparent thereafter. Al l of these events except for nuclear swelling were substantially blocked in MN9D/Bcl-X-L. At the later stage of cell death (< 32-36 hr), a specific cl eavage of Bax and fodrin appeared that was completely blocked by calpain in hibitor or by Bcl-X-L. Taken together, our data suggest that BCl-X-L preven ts etoposide-induced neuronal death by exerting its anticaspase and antical pain effect on cellular events after the formation of topoisomerase II-DNA cleavable complex that may not be a major contributor to cell death. (C) 20 01 Wiley-Liss, Inc.