Characterization of CPP32‐Like Protease Activity Following Apoptotic Challenge in SH‐SY5Y Neuroblastoma Cells
Characterization of CPP32‐Like Protease Activity Following Apoptotic Challenge in SH‐SY5Y Neuroblastoma Cells
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SH-SY5Y 神经母细胞瘤细胞凋亡挑战后 CPP32 样蛋白酶活性的表征
DOI：
10.1046/j.1471-4159.1997.68062328.x
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发表时间：
1997
影响因子：
4.7
通讯作者：
Kevin K. W. Wang
中科院分区：
文献类型：
--
作者：
R. Posmantur;K. McGinnis;Ravi Nadimpalli;R. Gilbertsen;Kevin K. W. Wang
摘要：我们研究了星形孢子素对人神经母细胞瘤细胞(SH-SY5Y)中白细胞介素1β转换酶(ICE)样蛋白(Caspase)的激活作用。乳酸脱氢酶释放的时间进程分析发现，在星状孢子素处理后，细胞死亡早在6h就显着增加，并持续到至少24h。用抗多聚腺苷二磷酸核糖聚合酶(抗PARP)和抗CPP32抗体进行Western印迹分析发现，CPP32(一种ICE同源物)在星状孢子素刺激后3h就发生了蛋白降解和PARP断裂。此外，CPP32底物乙酰-DEVD-7-氨基-4-甲基香豆素最早在3h被检测到，并在星状孢子素刺激后6h达到最大值，这表明CPP32样活性存在延迟和持续的时期。此外，我们还首次对细胞中的CPP32和PARP进行了免疫组织化学检测。CPP32在未经处理的SH-SY5Y细胞中的定位仅限于胞浆。星状孢子素攻击后，CPP32免疫荧光从细胞质凝聚到靠近质膜的区域。相反，在未处理的SH-SY5Y细胞中，PARP免疫荧光均匀分布在细胞核中，星形孢子素攻击被发现与浓缩的染色质有关。重要的是，PAN ICE抑制剂[carbobenzoxy‐Asp‐CH2OC(O)‐2，6‐dichlorobenzene]能够抑制星状孢子素攻击后乳酸脱氢酶的释放和PARP和CPP32的裂解，并改变PARP和CPP32的免疫组织化学染色模式。
Abstract: We characterized the activation of interleukin‐1β‐converting enzyme (ICE)‐like proteases (caspases) in human neuroblastoma cells (SH‐SY5Y) following challenge with staurosporine, an established agent known to induce apoptosis. Time course analyses of lactate dehydrogenase release detected a significant increase in cell death as early as 6 h that continued at least until 24 h following staurosporine treatment. Western blot analyses using anti‐poly(ADP‐ribose) polymerase (anti‐PARP) and anti‐CPP32 antibodies revealed proteolytic processing of CPP32 (an ICE homologue) as well as fragmentation of PARP as early as 3 h following staurosporine challenge. Furthermore, the hydrolysis of the CPP32 substrate acetyl‐DEVD‐7‐amido‐4‐methylcoumarin was detected as early as 3 h and became maximal at 6 h after staurosporine challenge, suggesting a delayed and sustained period of CPP32‐like activation. In addition, we used the first immunohistochemical examination of CPP32 and PARP in cells following an apoptotic challenge. The localization of CPP32 in untreated SH‐SY5Y cells was exclusively restricted to the cytoplasm. Following staurosporine challenge there was a condensing of CPP32 immunofluorescence from the cytoplasm to a region adjacent to the plasma membrane. In contrast, PARP immunofluorescence was evenly distributed in the nucleus in untreated SH‐SY5Y cells and on staurosporine challenge was found to be associated with condensed chromatin. It is important that a pan ICE inhibitor [carbobenzoxy‐Asp‐CH2OC(O)‐2,6‐dichlorobenzene] was able to attenuate lactate dehydrogenase release and PARP and CPP32 cleavage and altered immunohistochemical staining patterns for PARP and CPP32 following staurosporine challenge.
DOI：
10.1073/pnas.93.25.14486
发表时间：
1996-12-10
影响因子：
11.1
作者：
Srinivasula, SM;Ahmad, M;Alnemri, ES
通讯作者：
Alnemri, ES
影响因子：
11.2
作者：
T. Fernandes‐Alnemri;Atsushi Takahashi;R. Armstrong;J. Krebs;L. Fritz;K. Tomaselli;Lijuan Wang;Zailin Yu;C. Croce;Guy Salveson;W. Earnshaw;G. Litwack;E. Alnemri
通讯作者：
T. Fernandes‐Alnemri;Atsushi Takahashi;R. Armstrong;J. Krebs;L. Fritz;K. Tomaselli;Lijuan Wang;Zailin Yu;C. Croce;Guy Salveson;W. Earnshaw;G. Litwack;E. Alnemri
DOI：
10.1073/pnas.93.15.7464
发表时间：
1996-07-23
影响因子：
11.1
作者：
FernandesAlnemri, T;Armstrong, RC;Alnemri, ES
通讯作者：
Alnemri, ES