Genetic approaches for the identification of apoptotic components

Jun amino-terminal kinase (JNK) mediates a physiological stress signal that leads to cell death. However, the role of the JNK pathway in intrinsic cell death execution mechanisms is largely unknown. In a genetic screen for dominant suppressors of Reaper (Rpr)-induced cell death, we identified Drosophila chromosomal regions that contain genes which are homologous to apoptosis signal-regulating kinase (ASK1) and Drosophila tumor necrosis factor receptor-associated factor 1 (DTRAF1). We present evidence that the killer signal initiates the JNK pathway via proteasome-mediated degradation of Drosophila inhibitor of apoptosis protein 1 (DIAP1) to promote cell death.     

凝聚态Aβ 25～35可通过JNK/p38 MAPK途径诱导胎鼠皮层神经元Tau蛋白过度磷酸化

目的 探讨JNK/p38 MAPK在β淀粉样蛋白多肽片段25～35(Aβ25～35)诱导的阿尔茨海默病(AD)样胎鼠皮层神经元Tau蛋白过度磷酸化中的作用.方法 应用蛋白免疫印迹和免疫细胞化学染色的方法,观察Tau蛋白磷酸化和JNK/p38丝裂原活化的蛋白激酶(JNK/p38 MAPK)的表达情况.结果 凝聚态Aβ25～35(20μmol/L)作用于皮层神经元12h,Tau蛋白Ser396、Ser199/202、Thr205位点的磷酸化水平明显增高,同时JNK/p38 MAPK的总量及其活性形式-磷酸化JNK/p38 MAPK的蛋白表达水平也增加.结论 Aβ25～35可通过激活JNK/p38 MAPK使Tau蛋白的磷酸化水平增高.     

The mechanism of Ara-C-induced apoptosis of differentiating cerebellar granule neurons

Neurotoxicity is one of the side-effects of the therapeutically useful antitumour agent, Ara-C (or 1-beta-d-arabinofuranosyl-cytosine, cytarabine). This agent is also reported to induce cell death of cultured neurons. In this study, we show that Ara-C-induced death of differentiating rat cerebellar granule neurons is prevented by cycloheximide at concentrations corresponding to its action in preventing protein synthesis. The death is accompanied by cleavage of the caspase substrate poly ADP ribose polymerase (PARP) and c-Abl-dependent activation of the stress-activated protein kinases c-Jun N-terminal kinase and p38. However, c-Jun levels do not rise and the activation of the stress-activated protein kinases is not required for this form of neuronal death. Cyclin-dependent kinase (cdk) activity and inappropriate cell-cycle re-entry have been implicated in some forms of death in differentiated neurons. Here we show that Ara-C-induced death of cerebellar granule neurons is prevented by an inhibitor of cdk4, whereas inhibition of cdk1, -2 and -5 mimics the death, and non-cdk4/6 cdks are inhibited by Ara-C treatment. Cdk1 and -2 are dramatically down-regulated during neuronal differentiation, and neither Ara-C nor inhibition of these cdks induces death in mature neurons. This mechanism could also play a significant role in the neurotoxicity associated with the therapeutic use of Ara-C, as cdk levels can be upregulated in stressed neurons of adult brain. We propose that the balance between cdk4/6 and cdk1/2/5 activity may determine the survival of early differentiating neurons, and that DNA-damaging agents may induce neuronal death by inhibiting cdk1/2/5 under conditions which require these activities for survival.     

Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain

Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.     

bcl-xs基因对卵巢癌的作用及其凋亡机制的研究

目的 :研究全反式维甲酸 (ATRA)抑制视网膜母细胞瘤 (Rb)细胞生长的作用及信号转导机制。方法 :应用3 H 胸腺嘧啶掺入分析法观察ATRA对细胞生长的抑制作用 ,用流式细胞仪分析ATRA对Y79细胞周期的影响 ,用Westernblot分析c jun氨基末端激酶 (JNK)的磷酸化。 结果 :ATRA可明显地抑制Y79细胞的生长 ,10 -6mol·L-1ATRA处理 36h时 ,3 H 胸腺嘧啶掺入率下降达 4 0 % ,此条件下 ,Y79细胞被阻滞于G0 /G1期 ,并出现Sub G1峰 ;此生长抑制过程可被JNK的阻断剂Curcumin阻断 ;在此过程中 ,JNK被激活 ,磷酸化。结论 :ATRA可抑制Y79细胞生长 ,其过程是由磷酸化的JNK介导。提示 ,ATRA可能是一种潜在的抗视网膜母细胞瘤治疗药物     

促分裂原活化的蛋白激酶信号通路与能量代谢的关系

能量代谢每时每刻都伴随着物质代谢在生命体内发生。生物通过利用物质代谢产生的高能磷酸化合物以维持各项生命功能的运转,不同状态下的细胞通过利用各种物质通过不同的通路进行不同的代谢方式。促分裂原活化的蛋白激酶(MAPK)通路作为生命体里广泛存在的一类信号蛋白,影响着细胞、生物体的生命活动。MAPK通路在能量代谢的各个环节起着非常重要的作用。通过研究MAPK通路中的胞外信号调节激酶(ERK)通路、Jun激酶(JNK)通路、P38通路均与能量代谢有关的蛋白、靶点、关键酶、转运体,与三大营养物质能量代谢相结合,从而为能量代谢相关的细胞功能进一步研究铺垫,并为今后治疗肿瘤性疾病、感染性疾病、代谢性疾病等提供新思路。     

SP600125对D-氨基葡萄糖衍生物诱导Eca-109细胞凋亡的影响

目的:观察特异性JNK抑制剂[specificc-junNH2terminalproteinkinase(JNK)inhibitor]SP600125对D-氨基葡萄糖衍生物2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖{[2-(3-carboxy-1-oxopropyl)a-mino-2-deoxy-D-Glucose],COPADG}诱导Eca-109细胞凋亡的影响并探讨COPADG诱导Eca-109细胞凋亡的潜在分子机制。方法:体外培养Eca-109细胞,用COPADG及SP600125对细胞进行处理。细胞间接免疫荧光染色观察P-JNK蛋白表达的改变,倒置相差显微镜观察细胞形态学变化;MTT检测不同时间点的细胞活性;流式细胞术检测细胞凋亡率。结果:经SP600125处理后,COPADG诱导的Eca-109细胞P-JNK蛋白表达明显减弱,同时,COPADG诱导的Eca-109细胞凋亡率明显减低,细胞增殖抑制率下降明显,与COPADG单独作用组之间比较差异有统计学意义。结论:SP600125对D-氨基葡萄糖衍生物CO-PADG诱导Eca-109细胞凋亡具有抑制作用,并间接证明JNK信号转导通路在COPADG诱导Eca-109细胞凋亡过程中发挥着重要作用。     

Zearalenone Induces Apoptosis in Porcine Endometrial Stromal Cells through JNK Signaling Pathway Based on Endoplasmic Reticulum Stress

Zearalenone (ZEA) is an estrogen-like mycotoxin characterized mainly by reproductive toxicity, to which pigs are particularly sensitive. The aim of this study was to investigate the molecular mechanism of ZEA-induced apoptosis in porcine endometrial stromal cells (ESCs) by activating the JNK signaling pathway through endoplasmic reticulum stress (ERS). In this study, ESCs were exposed to ZEA, with the ERS inhibitor sodium 4-Phenylbutyrate (4-PBA) as a reference. The results showed that ZEA could damage cell structures, induce endoplasmic reticulum swelling and fragmentation, and decreased the ratio of live cells to dead cells significantly. In addition, ZEA could increase reactive oxygen species and Ca²⁺ levels; upregulate the expression of GRP78, CHOP, PERK, ASK1 and JNK; activate JNK phosphorylation and its high expression in the nucleus; upregulate the expression Caspase 3 and Caspase 9; and increase the Bax/Bcl-2 ratio, resulting in increased apoptosis. After 3 h of 4-PBA-pretreatment, ZEA was added for mixed culture, which showed that the inhibition of ERS could reduce the cytotoxicity of ZEA toward ESCs. Compared with the ZEA group, ERS inhibition increased cell viability; downregulated the expression of GRP78, CHOP, PERK, ASK1 and JNK; and decreased the nuclear level of p-JNK. The Bax/Bcl-2 ratio and the expression of Caspase 3 and Caspase 9 were downregulated, significantly alleviating apoptosis. These results demonstrate that ZEA can alter the morphology of ESCs, destroy their ultrastructure, and activate the JNK signaling via the ERS pathway, leading to apoptosis.