Gao Jian-Xin, Zhou Yu-Qin, Zhang Ru-Hua, Ma Xue-Lian, Liu Ke-Jing
Institute of Physiology, Medical School of Shandong University, Jinan 250012, China.
Sheng Li Xue Bao. 2005 Dec 25;57(6):755-60.
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 micromol/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 micromol/L) alone for 12 h was 29.03%, of the untreated controls. Both a broad-spectrum caspase inhibitor Z-VAD-FMK (50 mumol/L) and a specific caspase-3 inhibitor Z-DEVD-FMK (100 micromol/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 micromol/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 a-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.
罗可维汀是细胞周期蛋白依赖性激酶（cdks）cdc2/细胞周期蛋白B、cdk2/细胞周期蛋白A、cdk2/细胞周期蛋白E和cdk5/p35的特异性抑制剂。对罗可维汀的酶抑制特性和细胞效应的研究表明，它使细胞停滞在G(2)/M期和G(1)/S期，抑制哺乳动物细胞的增殖并诱导细胞死亡。然而，细胞死亡的特征以及这种cdk抑制剂杀死转化细胞的确切机制尚不清楚。我们之前研究发现罗可维汀可诱导有丝分裂的PC12细胞发生凋亡性死亡。本研究旨在确定罗可维汀诱导的死亡是否与凋亡途径中半胱天冬酶的特定元件有关。用hoechst 33342进行半胱天冬酶-3免疫荧光细胞化学双重染色的形态学数据表明，凋亡细胞核被鉴定为具有染色质凝聚和核碎裂的细胞核，并且在50 μmol/L罗可维汀处理4小时的PC12细胞中，半胱天冬酶-3活性p17亚基共存。与正常对照相比，半胱天冬酶-3阳性细胞数量显著增加至约42%（P<0.001）。MTT分析数据显示，单独用50 μmol/L罗可维汀处理12小时后的活细胞数量是未处理对照的29.03%。在存在罗可维汀的情况下，广谱半胱天冬酶抑制剂Z-VAD-FMK（50 μmol/L）和特异性半胱天冬酶-3抑制剂Z-DEVD-FMK（100 μmol/L）分别将活的PC12细胞增加至45.16%（Z-DEVD-FMK）和58.06%（Z-VAD-FMK）。非红细胞α-血影蛋白是一种细胞骨架蛋白，是半胱天冬酶-3半胱氨酸蛋白酶的底物。为了证实罗可维汀（50 μmol/L处理12小时）诱导的PC12细胞死亡中产生的半胱天冬酶-3的活性，使用小鼠抗非红细胞α-血影蛋白单克隆抗体通过蛋白质印迹法检测活化的半胱天冬酶-3特异性120 kDa血影蛋白降解产物（SBDP）。与正常对照相比，用罗可维汀处理的细胞溶质部分中对应于半胱天冬酶-3特异性SBDP水平的条带的平均相对密度显著增加（P<0.001）。这些结果表明，半胱天冬酶信号，尤其是半胱天冬酶-3信号对于罗可维汀诱发的增殖性PC12细胞凋亡性死亡的进展是必需的。
