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Caspase--3在roscovitine诱发PC12细胞凋亡中发挥重要作用

高建新, 周玉琴, 张茹华, 马雪莲, 刘克敬

山东大学医学院生理学研究所.山东，济南 250012

摘要

作者已证实周期蛋白激酶(cyclin--dependent kinases) cdk2、cdc2 和cdk5抑制剂roscovitine 诱导PC12 细胞凋亡。该实验应用caspase--3免疫细胞化学与hoechst 33342荧光化学双标、MTT比色法细胞活性测定和Western blot方法，研究了caspase--3在roscovitine所致PC12细胞凋亡中的作用。结果显示，roscovitine (50 #mu#mol/L) 处理PC12 细胞12 h, 细胞核染色质凝缩及核碎片形成，同时胞浆中出现caspase--3 阳性标志，caspase--3阳性细胞占细胞总数的42%。非特异性 caspases抑制剂Z--VAD--FMK (50 #mu#mol/L)和caspase--3特异性抑制剂Z-DEVD-FMK (100 #mu#mol/L)可部分降低roscovitine所致的细胞死亡，使细胞存活率分别由 29.03% (roscovitine) 增至58.06% (Z--VAD--FMK+roscovitine) 和45.16% (Z--DEVD--FMK+roscovitine) ；用单克隆non--erythroid #alpha#--spectrin 抗体检测roscovitine处理组细胞匀浆提取液，表明caspase--3 裂解的特异性spectrin 120 kDa蛋白产物较对照组显著增加。提示细胞凋亡成分caspases 参与roscovitine所致的细胞凋亡，其中caspase--3发挥重要作用。

关键词： 周期蛋白激酶; caspase-3; 凋亡; PC12 细胞; 细胞周期

Caspase-3 plays a required role in PC12 cell apoptotic death induced by roscovitine

Gao Jianxin, Zhou Yuqin, Zhang Ruhua, Ma Xuelian, Liu Kejing

Institute of Physiology, Medical School of Shandong University.Jinan 250012,Shandong

Abstract

Roscovitine is a specific inhibitor of cyclin-dependent kinases (cdks) cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35. The studies on the enzyme inhibitory properties and cellular effects of roscovitine revealed that it arrests cells in G_(2)/M and G_(1)/S phase, inhibits the proliferation of mammalian cells and induces cell death. However, the characteristics of cell death and exact mechanism by which this cdk inhibitor kills transformed cells are unknown. We previously investigated that the roscovitine induces apoptotic death of mitotic PC12 cells. The present study was to identify whether the roscovitine-induced death is related with the specific elements of caspases in pathway of apoptosis. The morphological data of caspase-3 immunofluorocytochemistry double staining with hoechst 33342 indicated that apoptotic nuclei were identified as nuclei with chromatin condensation and nuclear fragmentation, and that caspase-3 active p17 subunit co-existed in PC12 cells treated with roscovitine 50 μmol/L for 4 h. The number of the caspase-3 positive cells increased significantly to about 42%, as compared with the normal control (P<0.001). The data of MTT assay showed that the number of viable cells treated by roscovitine (50 #mu#mol/L) alone for 12 h was 29.03% of the untreated controls. Both a broad-spectrum caspase inhibitor Z-VAD-FMK (50 #mu#mol/L) and a specific caspase-3 inhibitor Z-DEVD-FMK (100 #mu#mol/L) increased viable PC12 cells to 45.16% (Z-DEVD-FMK) and 58.06% (Z-VAD-FMK), respectively, in the presence of roscovitine. Non-erythroid a-spectrin is a cytoskeleted protein that is a substrate of caspase-3 cysteine proteases. To confirm the activity of caspase-3 that produced in roscovitine (50 #mu#mol/L for 12 h)-induced PC12 cell death, activated caspase-3 specific 120 kDa spectrin breakdown products (SBDP) were detected by Western bloting using the mouse anti-non-erythroid #alpha#-spectrin monoclonal antibody. The mean relative density of bands corresponding to caspase-3 specific SBDP levels were significantly increased in the cytosolic fractions treated with roscovitine, as compared to the normal control (P<0.001). These results indicate that caspase signals, especially caspase-3 signal are necessary for the progression of proliferating PC12 cell apoptotic death evoked by roscovintine.

Key words: Cyclin-dependent kinases;Caspase-3;Apoptosis;PC12 cell;Cell cycle

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引用本文：

高建新, 周玉琴, 张茹华, 马雪莲, 刘克敬 . Caspase--3在roscovitine诱发PC12细胞凋亡中发挥重要作用[J]. 生理学报 2005; 57 (6): .

Gao Jianxin, Zhou Yuqin, Zhang Ruhua, Ma Xuelian, Liu Kejing . Caspase-3 plays a required role in PC12 cell apoptotic death induced by roscovitine . Acta Physiol Sin 2005; 57 (6): (in Chinese with English abstract).