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     

The Drosophila TNF ortholog Eiger: Emerging physiological roles and evolution of the TNF system

The TNF and TNFR superfamilies of proteins are conserved throughout evolution. The first invertebrate orthologs of TNF and TNFR, Eiger and Wengen, were identified in Drosophila, which enabled us to take advantage of its powerful genetics. Indeed, genetic studies on Eiger in the last decade have discovered their signaling mechanisms through activation of the JNK pathway and unveiled the role of Eiger-JNK signaling in a variety of cellular and tissue processes such as cell death, cell proliferation, tissue growth regulation, host defense, pain sensitization, and canalization. In this review, we will describe the in vivo signaling of Eiger and its physiological roles in fly development and homeostasis, and will discuss the evolution of the TNF/TNFR systems.     

Hyperbaric oxygen-stimulated proliferation and growth of osteoblasts may be mediated through the FGF-2/MEK/ERK 1/2/NF-κB and PKC/JNK pathways

We investigated whether the hyperbaric oxygen (O₂) could promote the proliferation of growth-arrested osteoblasts in vitro and the mechanisms involved in this process. Osteoblasts were exposed to different combinations of saturation and pressure of O₂ and evaluated at 3 and 7 days. Control cells were cultured under ambient O₂ and normal pressure [1 atmosphere (ATA)]; high-pressure group cells were treated with high pressure (2.5 ATA) twice daily; high-O₂ group cells were treated with a high concentration O₂ (50% O₂) twice daily; and high pressure plus high-O₂ group cells were treated with high pressure (2.5 ATA) and a high concentration O₂ (50% O₂) twice daily. Hyperbaric O₂ significantly promoted osteoblast proliferation and cell cycle progression after 3 days of treatment. Hyperbaric O₂ treatment stimulated significantly increased mRNA expression of fibroblast growth factor (FGF)-2 as well as protein expression levels of Akt, p70^S6K, phosphorylated ERK, nuclear factor (NF)-κB, protein kinase C (PKC)α, and phosphorylated c-Jun N-terminal kinase (JNK). Our findings indicate that high pressure and high O₂ saturation stimulates growth-arrested osteoblasts to proliferate. These findings suggest that the proliferative effects of hyperbaric O₂ on osteoblasts may contribute to the recruitment of osteoblasts at the fracture site. The FGF-2/MEK/ERK 1/2/Akt/p70^S6K/NF-κB and PKC/JNK pathways may be involved in mediating this process.     

Cytotoxicity of 15-Deoxy-��12,14-prostaglandin J2 through PPAR��-independent Pathway and the Involvement of the JNK and Akt Pathway in Renal Cell Carcinoma

Introduction: Agonists of peroxisome proliferator-activated receptor gamma (PPARγ) have been examined as chemopreventive and chemotherapeutic agents. The aim was to investigate the cytotoxicity and action mechanisms of 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂), one of endogenous ligands for PPARγ, in terms of PPARγ-dependency and the mitogen-activated protein kinase (MAPK) and Akt pathway in three human renal cell carcinoma (RCC)-derived cell lines.Methods: 786-O, Caki-2 and ACHN cells were used as human RCC-derived cell lines. Cell viability and caspase-3 activity was detected by fluorescent reagents, and chromatin-condensation was observed with a brightfield fluorescent microscope after staining cells with Hoechst33342. The expression levels of proteins were detected by Western blot analysis.Results: 15d-PGJ₂ showed cytotoxicity in dose-dependent manner. 15d-PGJ₂ induced chromatin-condensation and elevated caspase-3 activity, and the cell viability was restored by co-treatment with a pan-caspase inhibitor, Z-VAD-FMK, indicating the involvement of caspase-dependent apoptosis. The cytotoxicity was not impaired by a PPARγ inhibitor, GW9662, suggesting that 15d-PGJ₂ exerted the cytotoxicity in a PPARγ-independent manner. Some antioxidants rescued cells from cell death induced by 15d-PGJ₂, but some did not, suggesting that reactive oxygen species (ROS) did not contribute to the apoptosis. 15d-PGJ₂ also increased the expression levels of phospho-c-Jun N terminal kinase (JNK) in Caki-2 cells, and decreased those of phospho-Akt in 786-O cells, indicating that the JNK MAPK and the Akt pathways participated in the anticancer effects of 15d-PGJ₂ in some cell lines.Conclusion: 15d-PGJ₂ exerted cytotoxic effects accompanying caspase-dependent apoptosis, and this effect was elicited in a PPARγ-independent manner in three cell lines. In addition, the JNK MAPK and Akt pathway was involved in the cytotoxicity of 15d-PGJ₂ to some extent in some cell line. Therefore, our study showed the 15d-PGJ₂ to potentially be an interesting approach for RCC treatment.     

Mutations in the transcription factor FOXO1 mimic positive selection signals to promote germinal center B cell expansion and lymphomagenesis

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JNK信号通路在小鼠术后肠麻痹发病机制中的作用

目的 探讨JNK信号通路在小鼠术后肠麻痹（POI）发病机制中的作用。方法 将野生型C57/BL6小鼠（WT）及同品系的JNK-/-小鼠均随机分成假手术组（Sham组，n=6）和肠麻痹组（POI组，n=6）。采用经典小肠操作方法诱导POI模型，术后24h给小鼠碳末灌胃，20min后麻醉小鼠，开腹取小肠评估肠动力，取回肠评估组织学改变，检测髓过氧化物酶（MPO）、IL-1β、IL-6水平及Claudin-2蛋白表达。结果 与Sham组小鼠相比，无论WT或JNK-/-小鼠其POI组的小肠排推率（分别为21%与33%）均明显降低（P=0.034及P=0.045，均P<0.05），小肠组织MPO活性水平（分别为0.608U/g与0.433U/g）明显升高（均P<0.05）；与WT小鼠POI组比较，JNK-/-小鼠POI组的小肠运动功能及其组织病理变化有所改善，炎症介质如MPO、IL-1β及IL-6水平均有明显降低（均P<0.05），小肠Claudin-2蛋白表达也降低（P<0.01）。结论 JNK基因敲除减轻小鼠肠道炎症反应、改善POI，表明JNK信号通路参与POI的发病过程。