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 suppressor gene product p53, and the ICE/ced-3-related proteases (caspases) have all been implicated in programmed cell death in a wide variety of cell types. However, their roles in radiation-induced neuronal cell death are poorly understood. In order to further elucidate the molecular mechanisms underlying radiation-induced neuronal cell death, we have examined the ability of ionizing radiation to induce cell death in primary cultured hippocampal neurons obtained from wild-type, p53-deficient and Bax-deficient newborn mice. Survival in neuronal cultures 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 contrast, neuronal survival in irradiated cultures derived from p53-deficient or Bax-deficient mice was equivalent to that observed in control, nonirradiated cultures. Western blot analyses indicated that neuronal p53 protein levels increased after irradiation in wild-type cells. However, Bax protein levels did not change, indicating that other mechanisms exist for regulating Bax activity. Adenovirus-mediated overexpression of p53 also caused neuronal cell death without increasing Bax protein levels. Irradiation resulted in a significant induction in caspase activity, as measured by increased cleavage of fluorogenic caspase substrates. However, specific inhibitors of caspase activity (zVAD-fmk, zDEVD-fmk and BAF) failed to protect postnatal hippocampal neurons from radiation-induced cell death. Staurosporine (a potent inducer of apoptosis in many cell types) effectively induced neuronal cell death in wild-type, p53-deficient and Bax-deficient hippocampal neurons, indicating that all were competent to undergo programmed cell death. These results demonstrate that both p53 and Bax are necessary for radiation-induced cell death in postnatal cultured hippocampal neurons. The fact that cell death occurred despite caspase inhibition suggests that radiation-induced neuronal cell death may occur in a caspase-independent manner.
Johnson
M D
MD
Department of Neurological Surgery, University of Washington School of Medicine, Seattle 98195-6470, USA.
Xiang
H
H
London
S
S
Kinoshita
Y
Y
Knudson
M
M
Mayberg
M
M
Korsmeyer
S J
SJ
Morrison
R S
RS
D000818:Animals; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism / Q000528:radiation effects*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000502:physiology / Q000528:radiation effects*; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000528:radiation effects*; D019253:Proto-Oncogene Proteins c-bcl-2; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D019311:Staurosporine / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism / Q000528:radiation effects*; D051028:bcl-2-Associated X Protein
C494827:Bax protein, mouse; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011838:Radiation-Sensitizing Agents; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D020169:Caspases; D019311:Staurosporine