Recent evidence demonstrates that the proto-oncogene product, Bcl-2 can protect cells from a variety of cell death-inducing stimuli. Because previous studies have demonstrated that protein kinase (PK) pathways may be involved in the regulation of cell death, we tested various PK inhibitors for their effects on cell death in a dopaminergic neuronal cell line, MN9D, as well as the potential of Bcl-2 family members and structural mutants to block this process. Cells expressing either human Bcl-2 (MN9D/Bcl-2), or neomycin (MhT9D/Neo; control cells) were treated with either staurosporine (0.25-2 μM) or trifluoperazine (10-100 μM). In control MN9D/Neo cells, both reagents led to a dose-dependent cell death with morphological features of apoptosis. Overexpression of Bcl-2 rescued cells from staurosporine-induced but not trifluoperazine-induced apoptotic cell death. Cell death induced by the specific PKC inhibitor, calphostin C was also significantly attenuated in MN9D/Bcl-2 cells indicating that a PKC pathway represents one mechanism by which Bcl-2 prevents staurosporine-induced cell death. Similarly, the Bcl-2 family member, Bcl-X(L) also blocked staurosporine-induced cell death in MN9D cells whereas overexpression of Bcl-X(S) or Bax did not. Finally, staurosporine-induced cell death was still blocked by the expression of clones encoding mutations in the Bcl-2 homology domains, BH1 and BH2, as well as C-terminally truncated Bcl-2. These data suggest that in the staurosporine-mediated cell death model Bcl-2 is not heterodimerizing to related proteins through these highly conserved structural domains nor does it need to be membrane-anchored. Thus, in this paradigm, either Bcl-2 functions as a homodimer or essential sequences lie outside of the BH1 or BH2 domains.
Bibliographical noteFunding Information:
We would like to thank Drs. A. Heller, L. Won and P. Hoffman for providing us with the MN9D cell line, as well as helpful discussions. This work was supported in part by DA08818, MH31302 and the Korean Science Foundation 95-0403-94-3 (to Y.J.O.).
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cellular and Molecular Neuroscience