Sequential cleavage of poly(ADP-ribose)polymerase and appearance of a small Bax-immunoreactive protein are blocked by Bcl-XL and caspase inhibitors during staurosporine-induced dopaminergic neuronal apoptosis

To assess the role of Bcl-X-L and its splice derivative, Bcl-X-S, in staurosporine-induced cell death, we used a dopaminergic cell line, MN9D, transfected with bcl-x(L) (MN9D/Bcl-X-L), bcl-x(S) (MN9D/Bcl-X-S), or control vector (MN9D/Neo). Only 8.6% of MN9D/Neo cells survived after 24 h of 1 mu M staurosporine treatment. Caspase activity was implicated because a caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk), attenuated staurosporine-induced cell death. Bcl-X-L rescued MN9D cells from death (89.4% viable cells), whereas Bcl-X-S had little or no effect. Bcl-X-L prevented morphologically apoptotic changes as well as cleavage of poly(ADP-ribose)polymerase (PARP) induced by staurosporine. It is interesting that a small Bax-immunoreactive protein appeared 4-8 h after PARP cleavage in MN9D/Neo cells. The appearance of the small Bax-immunoreactive protein, however, may be cell type-specific as it was not observed in PC12 cells after staurosporine treatment. The sequential cleavage of PARP and the appearance of the small Bax-immunoreactive protein in MN9D cells were blocked either by Z-VAD-fmk or by Bcl-X-L. Thus, our present study suggests that Bcl-X-L but not Bcl-X-S prevents staurosporine-induced apoptosis by inhibiting the caspase activation that may be directly or indirectly responsible for the appearance of the small Bax-immunoreactive protein in some types of neurons.