Bax, a member of the Bcl-2 gene family, is known to promote apoptosis in many cases but to block cell death under certain conditions. To investigate the potential role of Bax in 6-hydroxydopamine (6-OHDA)-induced cell death, we first established and characterized a dopaminergic neuronal cell line (MN9D) stably overexpressing hemagglutinin epitope-tagged Bax (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 microscopy and agarose gel analysis. Overexpression of HA-Bax in MN9D cells was shown to attenuate 6-OHDA-induced cell death as determined by the MTT reduction assay and agarose gel analysis for DNA fragmentation. Western blot analysis 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 attenuate 6-OHDA-induced cell death. Interestingly, cell death induced by hydrogen peroxide (0.25-2.0 mM) was significantly accelerated, whereas the rate of cell death induced by menadione (10-50 μM) was not affected in MN9D/HA-Bax cells. Thus, our present data suggest that the functionally diverse roles of Bax may be determined by the type of stress applied to the cell.
Bibliographical noteFunding Information:
We gratefully acknowledge Drs. A. Heller and Lisa Won for providing us with the MN9D cell line (University of Chicago). We also thank Drs. J. C. Reed and H. Zha for providing the HA-Bax expression plasmid (Burnham Institute) and Ms. S. J. Joo for sequencing HA-Bax cDNA. This work was supported by the Genetic Engineering Grants from the Ministry of Education, KOSEF 95-0403-94-3 and STEPI (to Y.J.O.) and MH45330 and National Parkinson Foundation, Inc. (to K.L.O.).
All Science Journal Classification (ASJC) codes
- Developmental Neuroscience