Johnson M D, Xiang H, London S, Kinoshita Y, Knudson M, Mayberg M, Korsmeyer S J, Morrison R S
Department of Neurological Surgery, University of Washington School of Medicine, Seattle 98195-6470, USA.
J Neurosci Res. 1998 Dec 15;54(6):721-33. doi: 10.1002/(SICI)1097-4547(19981215)54:6<721::AID-JNR1>3.0.CO;2-1.
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.
Bax（bcl - 2基因家族中促进细胞死亡的成员）、肿瘤抑制基因产物p53以及与ICE/ced - 3相关的蛋白酶（半胱天冬酶）均与多种细胞类型的程序性细胞死亡有关。然而，它们在辐射诱导的神经元细胞死亡中的作用却知之甚少。为了进一步阐明辐射诱导神经元细胞死亡的分子机制，我们研究了电离辐射诱导从野生型、p53基因缺陷型和Bax基因缺陷型新生小鼠获取的原代培养海马神经元细胞死亡的能力。单次给予10 Gy至30 Gy剂量的电离辐射24小时后，源自野生型小鼠的神经元培养物中的细胞存活率呈剂量依赖性下降。相比之下，源自p53基因缺陷型或Bax基因缺陷型小鼠的受辐照培养物中的神经元存活率与未受辐照的对照培养物中的存活率相当。蛋白质免疫印迹分析表明，野生型细胞受辐照后神经元p53蛋白水平升高。然而，Bax蛋白水平未发生变化，这表明存在其他调节Bax活性的机制。腺病毒介导的p53过表达也导致神经元细胞死亡，且未增加Bax蛋白水平。通过荧光半胱天冬酶底物切割增加来衡量，辐照导致半胱天冬酶活性显著诱导。然而，半胱天冬酶活性的特异性抑制剂（zVAD - fmk、zDEVD - fmk和BAF）未能保护新生海马神经元免受辐射诱导的细胞死亡。星形孢菌素（许多细胞类型中一种有效的凋亡诱导剂）有效地诱导野生型、p53基因缺陷型和Bax基因缺陷型海马神经元细胞死亡，表明所有这些神经元都能够经历程序性细胞死亡。这些结果表明，p53和Bax对于出生后培养的海马神经元中辐射诱导的细胞死亡都是必需的。尽管半胱天冬酶受到抑制但细胞死亡仍发生这一事实表明，辐射诱导的神经元细胞死亡可能以不依赖半胱天冬酶的方式发生。
