Overexpression of HA-Bax but not Bcl-2 or Bcl-X-L attenuates 6-hydroxydopamine-induced neuronal apoptosis

Bar, a member of the Bcl-2 gene family, is known to promote apoptosis in ma ny cases but to block cell death under certain conditions. To investigate t he potential role of Bar in B-hydroxydopamine (6-OHDA)induced cell death, w e first established and characterized a dopaminergic neuronal cell Line (MN 9D) stably overexpressing hemagglutinin epitope-tagged Bar (MN9D/HA-Bax) as well as control clones (MN9D/Neo). Treatment of MN9D/Neo cells with 6-OHDA induced typical apoptotic cell death accompanied by shrinkage of the cell, nuclear condensation, and DNA fragmentation as demonstrated by light micro scopy and agarose gel analysis. Overexpression of HA-Bar in MN9D cells was shown to attenuate 6-OHDA-induced cell death as determined by the MTT reduc tion assay and agarose gel analysis for DNAfragmentation. Western blot anal ysis revealed that cleavage of poly(ADP-ribose)polymerase induced by 6-OHDA was attenuated in MN9D/HA-Bax cells, In contrast, overexpression of a well -known cell death-inhibiting protein such as Bcl-2 or Bcl-X-L did not atten uate 6-OHDA-induced cell death. Interestingly, cell death induced by hydrog en peroxide (0.25-2.0 mM) was significantly accelerated, whereas the rate o f cell death induced by menadione (10-50 mu M) was not affected in MN9D/HA- Bax cells. Thus, our present data suggest that the functionally diverse rol es of Bar may be determined by the type of stress applied to the cell. (C) 1998 Academic Press.