Evidence for involvement of Bax and p53, but not caspases, in radiation-induced cell death of cultured postnatal hippocampal neurons

Abstract

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.

Neural precursor cells possess multiple p53-dependent apoptotic pathways

“…36 Of the proapoptotic members of the Bcl-2 family, Bax is considered to be the most significant mediator of mitochondria-dependent apoptosis in neurons. 29,30 The contribution of Bak to neuronal apoptosis depends on the cell type and apoptotic stimulus. 28 Bax deficiency alone partially protects primary telencephalic NPCs against DNA damage-induced death; however, dual Bax and Bak deficiency provides an even greater protective effect against this apoptotic stimulus.…”

“…Interestingly, in certain neuronal populations, STS does not appear to induce a p53-or Bax-dependent apoptosis. 30 Like NPCs, SH-SY5Y neuroblastoma cells undergo a transcription-independent death in response to STS that is prevented by inactivation of caspase-9. 16 However, STS-induced apoptosis of SH-SY5Y cells is unaffected by p53 inactivation, 16 further highlighting the remarkable cell-type specificity of apoptosis regulation.…”

“…The proapoptotic molecules Bax and Bak play overlapping functions in the regulation of apoptosis, and it appears that the significance of either molecule is highly dependent on cell type. 11,[28][29][30] We reasoned that either Bax or Bak might be critical for the activation of the intrinsic apoptotic pathway in NPCs. To test this hypothesis, we crossed mice that were heterozygous for both bax and bak and prepared FGF2-expanded NPCs from E13 embryos.…”

Apoptosis without caspases: an inefficient molecular guillotine?

“…82,83 Similarly, Z-VAD.fmk inhibited nuclear damage of target cells induced by exocytotic granules of cytotoxic T cells but did not prevent target cell lysis (as measured by Cr 51 release). 84,85 More recent examples include apoptosis induced by the GD3 ganglioside, 86 class I MHC antibodies, 87 puromycin, 88 polyamine analogues, 89 CD-2 and staurosporine, 90,91 intracellular acidification, 92 the retinoid AHPN, 93 E4orf4, a novel adenovirus death factor, 94 irradiation, 95,96 VP-16, dexamethasone and actinomycin D, 97 ± 99 PML, 100 and nitric oxide (NO). 101 Even some aspects of Fas-induced apoptosis (when triggered with the adapter FADD) may occur caspaseindependently although in this case necrotic morphologies were observed as well.…”

Regulation of AIF expression by p53

“…7 However, in the presence of caspase inhibitors, p53 can also act via a delayed onset caspase-independent mechanism, at least in some cell models. 8,9 Sequence-specific binding of wild-type p53 to p53 response elements (p53REs) within the DNA is essential for its role as a tumor suppressor. The transactivation of different target genes differs: 10 some genes require high p53 levels, which are induced only upon exposure to p53-activating stress, whereas others are already upregulated by endogenous p53 levels.…”