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

1)我们已经建立了几个局灶黏附激酶（FAK）转染HL-60 （HL-60/FAK）细胞，这些细胞对氧化应激诱导的凋亡具有抗性。我们观察到HL-60/FAK细胞的增殖速度比载体转染（HL-60/Vect）细胞快得多。这一观察结果促使我们研究HL-60/FAK细胞增强细胞增殖的机制。由于PKC抑制剂chelerythrine或PI3-kinase抑制剂ly294002可有效抑制细胞增殖，因此PKC和PI-3-kinase通路均参与细胞增殖。因为在HL-60/FAK细胞中，cyclin D3的表达特别突出，PKCα、β和η亚型被激活，并与FAK直接相关。因此，我们假设FAK激活PKC和pi3激酶- akt通路，从而显著诱导cyclin D3表达和CDK活性。2)利用细胞因子趋化因子和凋亡芯片进行cDNA芯片筛选，鉴定相关分子。我们发现H_2O_2处理后HL-60/FAK细胞谷胱甘肽过氧化物酶（GPx） mRNA表达降低，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细胞中谷胱甘肽还原酶（GRe）、磷脂氢过氧化物谷胱甘肽过氧化物酶（PHGPx）的基础活性和mRNA表达均显著升高。HL-60/FAK细胞中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，导致氧化应激的抗凋亡状态。

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.