EVIDENCE FOR INVOLVEMENT OF BAX AND P53, BUT NOT CASPASES, IN RADIATION-INDUCED CELL-DEATH OF CULTURED POSTNATAL HIPPOCAMPAL-NEURONS

Bax (a death-promoting member of the bcl-2 gene family), the tumor sup pressor gene product p53, and the ICE/ced-3-related proteases (caspase s) have all been implicated in programmed cell death in a wide variety of cell types. However, their roles in radiation-induced neuronal cel l death are poorly understood. In order to further elucidate the molec ular mechanisms underlying radiation-induced neuronal cell death, we h ave examined the ability of ionizing radiation to induce cell death in primary cultured hippocampal neurons obtained from wild-type, p53-def icient and Bax-deficient ne newborn mice. Survival in neuronal culture s derived from wild-type mice decreased in a dose-dependent manner 24 hr after a single 10 Gy to 30 Gy dose of ionizing radiation. In contra st, neuronal survival in irradiated cultures derived from p53-deficien t or Bax-deficient mice was equivalent to that observed in control, no nirradiated cultures, Western blot analyses indicated that neuronal p5 3 protein levels increased after irradiation in wild-type cells, Howev er, Bax protein levels did not change, indicating that other mechanism s exist for regulating Bas activity, Adenovirus-mediated overexpressio n of p53 also caused neuronal cell death without increasing Bax protei n levels. Irradiation resulted in a significant induction in caspase a ctivity, as measured by increased cleavage of fluorogenic caspase subs trates. However specific inhibitors of caspase activity (zVAD-fmk, zDE VD-fmk and BAF) failed to protect postnatal hippocampal neurons from r adiation-induced cell death. Staurosporine (a potent inducer of apopto sis in many cell types) effectively induced neuronal cell death in wil d-type, p53-deficient and Bax-deficient hippocampal neurons, indicatin g that all were competent to undergo programmed cell death. These resu lts demonstrate that both p53 and Bax are necessary for radiation-indu ced cell death in postnatal cultured hippocampal neurons. The fact tha t cell death occurred despite caspase inhibition suggests that radiati on-induced neuronal cell death may occur in a caspase-independent mann er, J. Neurosci. Res. 54:721-733, 1998. (C) 1998 Wiley-Liss, Inc.