STUDY ON THE NEW ANTI-APOPTOTIC MOLECULES ANALYZED BY THE DNA MICROARRAY METHODS

DNA微阵列方法分析新型抗凋亡分子的研究

• 批准号: 14572066
• 负责人: KASAHARA Tadashi
• 金额: $ 2.24万
• 依托单位: KYORITSU WOMEN'S UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572066/
• 关键词: focal adhesion kinase (FAK); oxidative-stress; DNA microarray; PI3-kinase; protein kinase C; anti-apoptosis; glutathione peroxidase; シグナル分子; HL-60; 2次元電気泳動; レドックス制御; 接着斑キナーゼ; DNAマイクロアレー; タンパク質のプロファイリング

1) We have established several focal adhesion kinase (FAK)-transfected HL-60 (HL-60/FAK) cells which become resistant to oxidative stress-induced apoptosis. We observed that HL-60/FAK cells proliferate much faster than vector-transfected (HL-60/Vect) cells. This observation prompted us to investigate the mechanism how HL-60/FAK cells augment cell proliferation. Since a PKC inhibitor, chelerythrine or a PI3-kinase inhibitor, LY 294002 suppressed cell proliferation effectively, both PKC and PI-3-kinase pathways are presumed to be involved in the cell proliferation. Since cyclin D3 expression was particularly prominent and PKCα, β, and η isoforms were activated and directly associated with FAK in HL-60/FAK cells. We thus assumed that FAK activates PKC and PI3-kinase-Akt pathway, which resulted in marked induction of cyclin D3 expression and CDK activity.2) We performed cDNA microarray screening using cytokine-chemokine and apoptosis-chip to identify responsible molecules. We found that glutathione peroxidase (GPx) mRNA was decreased and lipid peroxidation was suppressed after treatment with H_2O_2 in HL-60/FAK cells. In addition, HL-60/FAK cells have higher basal ROS levels. Basal activity and mRNA expression of GSH reductase (GRe), phospholipid hydroperoxide glutathione peroxidase (PHGPx) were markedly elevated in HL-60/FAK cells. In contrast, GPx and catalase levels were decreased in HL-60/FAK cells. Thus, we suggested that FAK upregulates antioxidant enzymes and suppresses lipid peroxidation, resulting in the anti-apoptotic state for oxidative stress.

1)我们建立了几个FAK转染型HL-60(HL-60/FAK)细胞，这些细胞对氧化应激诱导的细胞凋亡具有抵抗力。我们观察到HL-60/FAK细胞的增殖速度明显快于载体转染组(HL-60/Vect)。这一观察结果促使我们研究HL-60/FAK细胞促进细胞增殖的机制。由于蛋白激酶C抑制剂白屈菜红碱或PI3-激酶抑制剂，LY 294002有效地抑制了细胞的增殖，因此，蛋白激酶C和PI-3-激酶通路都被认为参与了细胞的增殖。由于在HL-60/FAK细胞中Cyclin D3的表达尤为突出，并且PKC、α，β和η亚型被激活，并与FAK直接相关。因此，我们推测FAK激活了PKC和PI3-K-Akt通路，导致了细胞周期蛋白D3的表达和CDK活性的显著诱导。2)我们利用细胞因子-趋化因子和凋亡芯片进行了基因芯片筛选，以确定相关的分子。我们发现，H_2O_2处理HL-60/FAK细胞后，细胞内谷胱甘肽过氧化物酶(GPX)基因表达减少，脂质过氧化作用受到抑制。此外，HL-60/FAK细胞具有较高的基础ROS水平。HL-60/FAK细胞GSH还原酶(GRE)、磷脂过氧化氢谷胱甘肽过氧化物酶(PHGPx)的基础活性和mRNA表达均显著升高。与之相比，HL-60/FAK细胞中的GPX和过氧化氢酶水平降低。因此，我们认为FAK上调抗氧化酶，抑制脂质过氧化，从而导致氧化应激的抗凋亡状态。

项目成果

Watanabe H, Adachi R, Hirayama A, Kasahara T, Suzuki K.: "Triphenyltin enhances the neutrophilic differentiation of promyelocytic HL-60 cells."Biochem Biophys Res Commun. 306(1). 26-31 (2003)

Watanabe H、Adachi R、Hirayama A、Kasahara T、Suzuki K.：“三苯基锡增强早幼粒细胞 HL-60 细胞的中性粒细胞分化。”Biochem Biophys Res Commun。

Sakurai S, Sonoda Y, Kasahara T: "Mutated focal adhesion kinase induces apoptosis in a human glioma cell T98G"Biochem Biophys Res Commun. 293(1). 147-181 (2002)

Sakurai S、Sonoda Y、Kasahara T：“突变的粘着斑激酶诱导人神经胶质瘤细胞 T98G 细胞凋亡”Biochem Biophys Res Commun。

Kasahara T, Yokota E, Sonoda Y, et al.: "Antiapoptotic action of focal adhesion kinase (FAK) against ionizing radiation."Antioxid Redox Signal. 4(3). 491-499 (2002)

Kasahara T、Yokota E、Sonoda Y 等人：“粘着斑激酶 (FAK) 对电离辐射的抗凋亡作用。”抗氧化氧化还原信号。

Funakoshi-Tago M, Sonoda Y, Kasahara T et al.: "TRAF6 and C-SRC induce synergistic AP-1 activation via P13-Kinase-AKT-JNK Pathway"Eur J Biochem. 270(6). 1257-1268 (2003)

Funakoshi-Tago M、Sonoda Y、Kasahara T 等人：“TRAF6 和 C-SRC 通过 P13-激酶-AKT-JNK 途径诱导 AP-1 协同激活”Eur J Biochem。

Matsui S, Matsumoto S, Kasahara T et al.: "LIM Kinase 1 modulates opsonized zymosan-triggered activation of macrophage-like U937 cells"J Biol Chem. 277(1). 544-549 (2002)

Matsui S、Matsumoto S、Kasahara T 等人：“LIM 激酶 1 调节调理酶聚糖触发的巨噬细胞样 U937 细胞的激活”J Biol Chem。