Apoptosis mediated by p53 in rat neural AF5 cells following treatment with hydrogen peroxide and staurosporine
Apoptosis mediated by p53 in rat neural AF5 cells following treatment with hydrogen peroxide and staurosporine
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DOI：
10.1016/j.brainres.2006.07.024
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发表时间：
2006-09-27
期刊：
影响因子：
2.9
通讯作者：
Merrick, B. Alex
中科院分区：
文献类型：
--
作者：
McNeill-Blue, Charlesene;Wetmore, Barbara A.;Merrick, B. Alex
用缺少p53失活结构域的截短的SV 40 LT载体使来自胎鼠中脑组织的AF 5神经细胞永生化，以保持具有p53应答表型的长期培养物。本研究探讨了p53在产生程序性细胞死亡增殖AFS神经细胞暴露后，过氧化氢（H2 O2）和激酶抑制剂staurosporine（STSP）的功能。将AF 5细胞暴露于0-800 mM H2 O2和0-1000 nM STSP的浓度依赖性暴露显示MTS细胞活力测定的细胞毒性增加。通过subG（1）DNA和膜联蛋白V流式细胞术分析以及碘化丙啶、抗膜联蛋白V和DAPI的细胞免疫荧光染色证明，在400 mM H2 O2下发生细胞凋亡。DNA片段化，caspase-3/7活性和细胞色素c释放到胞质溶胶中也证实了H2 O2介导的凋亡事件。p53蛋白水平增加超过24小时的H2 O2在协调的方式与mdm 2的表达。通过升高的Ser 15磷酸化、增加的荧光素酶p53报告基因活性和下游p53靶点p21（waf 1）和凋亡蛋白bax、Noxa和p53 A的上调，证实了H2 O2对pS3的激活。STSP暴露产生的细胞凋亡表现为DNA片段化，caspase-3/7活性，细胞色素c的释放和超过24小时，伴随着持续增加的p53和Ser 15磷酸化，上升p21（wafl）和bax和p53报告活性的瞬时增加，但没有膜联蛋白V结合。这些研究结果表明，AF 5细胞发生凋亡响应H2 O2介导的氧化应激和信号通路中断STSP，因此将是有用的研究相关的p53依赖性神经元细胞死亡和神经元变性。由爱思唯尔公司出版
AF5 neural cells derived from fetal rat mesencephalic tissue were immortalized with a truncated SV40 LT vector lacking the p53-inactivating domain to maintain long-term cultures with a p53-responsive phenotype. This study examined p53 function in producing programmed cell death in propagating AFS neural cells after exposure to hydrogen peroxide (H2O2) and the kinase inhibitor staurosporine (STSP). Concentration-dependent exposure of AF5 cells to 0-800 mM H2O2 and STSP at 0-1000 nM revealed increasing cytotoxicity from MTS cell viability assays. Apoptosis occurred at 400 mM H2O2 as evidenced by subG(1) DNA and Annexin V flow cytometry analyses and cellular immunofluorescence staining with propidium iodide, anti-Annexin V and DAPI. DNA fragmentation, caspase-3/7 activity and cytochrome c release into cytosol also confirmed H2O2-mediated apoptotic events. p53 protein levels were increased over 24 h by H2O2 in a coordinated fashion with mdm2 expression. pS3 activation by H2O2 was evidenced by elevated Ser15 phosphorylation, increased luciferase p53 reporter activity and upregulation of the downstream p53 targets p21(waf1) and apoptotic proteins, bax, Noxa and PUMA. STSP exposure produced apoptosis demonstrated by DNA fragmentation, caspase-3/7 activity, cytochrome c release and over 24 h was accompanied by sustained increase in p53 and Ser15 phosphorylation, rise in p21(wafl) and bax and a transient increase in p53 reporter activity but without Annexin V binding. These findings demonstrate that AF5 cells undergo apoptosis in response to H2O2-mediated oxidative stress and signal pathway disruption by STSP that therefore would be useful in studies related to p53-dependent neuronal cell death and neuro degeneration. Published by Elsevier B.V.