REGIONS OUTSIDE OF THE BCL-2 HOMOLOGY DOMAINS, BH1 AND BH2 PROTECT A DOPAMINERGIC NEURONAL CELL-LINE FROM STAUROSPORINE-INDUCED CELL-DEATH

Recent evidence demonstrates that the proto-oncogene product, Bcl-2 ca n 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 huma n Bcl-2 (MN9D/Bcl-2), or neomycin (MN9D/Neo; control cells) were treat ed with either staurosporine (0.25-2 mu M) or trifluoperazine (10-100 mu M). In control MN9D/Neo cells, both reagents led to a dose-dependen t cell death with morphological features of apoptosis. Overexpression of Bcl-2 rescued cells from staurosporine-induced but not trifluoperaz ine-induced apoptotic cell death. Cell death induced by the specific P KC inhibitor, calphostin C was also significantly attenuated in MN9D/B cl-2 cells indicating that a PKC pathway represents one mechanism by w hich Bcl-2 prevents staurosporine-induced cell death. Similarly, the B cl-2 family member, Bcl-X-L also blocked staurosporine-induced cell de ath in MN9D cells whereas overexpression of Bcl-X-S or Bax did not. Fi nally, staurosporine-induced cell death was still blocked by the expre ssion of clones encoding mutations in the Bcl-2 homology domains, BH1 and BH2, as well as C-terminally truncated Bcl-2. These data suggest t hat in the staurosporine-mediated cell death model Bcl-2 is not hetero dimerizing to related proteins through these highly conserved structur al domains nor does it need to be membrane-anchored. Thus, in this par adigm, either Bcl-2 functions as a homodimer or essential sequences li e outside of the BH1 or BH2 domains. (C) 1997 Elsevier Science B.V.