Analysis of the effect for proliferation & therapeutic sensitivity according to specific binding protein for each hypoxia-inducible factor(HIF) 1α & 2α

根据每种缺氧诱导因子 (HIF) 1α 和 2α 的特异性结合蛋白分析对增殖和治疗敏感性的影响

• 批准号: 21592054
• 负责人: KONDO Keiichi
• 金额: $ 2.91万
• 依托单位: Yokohama City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2009
• 资助国家: 日本
• 起止时间: 2009 至 2011
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21592054/
• 关键词: HIF; 結合蛋白; 低酸素環境; 腎細胞癌; 治療感受性

(Introduction) Renal cell carcinoma specimens show variable staining pattern of hypoxia-inducible factor(HIF) 1α & 2α. HIF1α & 2αhave high homology, but different function. So far, it has not been clear what kind of function these proteins have specifically. To clarify this point, we search specific binding proteins for each HIFα.(Materials & Methods) We extract proteins from renal cell carcinoma cell line ACHN & SN12C, which show normal von Hippel-Lindau(VHL) protein function and express both HIFαs under hypoxic condition, and immunoprecipitated them to find specific binding proteins with HIFα specific antibodies. Co-immunoprecipetated proteins were dissolved by SDS-PAGE, and expression level compared by silver staining.(Results & future plans) We could detect several candidate bands, which shows stronger expression compared with samples co-precipitated by control antibody. Next we tried to dissolve them by 2D-electrophoresis and picked up candidate spots to do mass-spectrometry analysis. But amount of these proteins were so little and we could not get reliable results, so far. It is unable to amplify these proteins in cancer cells, because we had not yet identified. Now we try to enhance affinity between HIFαs & these proteins. We change culture conditions such as oxygen and/or glucose concentrations to optimize each HIFα expression, respectively and check the affinity under such conditions.

肾细胞癌组织中缺氧诱导因子（hypoxia-inducible factor，HIF）1α和2α的表达具有多样性。HIF 1 α和HIF 2α具有高度同源性，但功能不同。到目前为止，还不清楚这些蛋白质具体具有什么样的功能。为了阐明这一点，我们寻找每个HIFα的特异性结合蛋白。（材料与方法）从低氧条件下表达HIFα和VHL蛋白的肾癌细胞系ACHN和SN 12 C中提取蛋白质，免疫沉淀HIFα特异性抗体，寻找特异性结合蛋白。通过SDS-PAGE溶解共免疫沉淀的蛋白，并通过银染色比较表达水平。我们可以检测到几个候选条带，与对照抗体共沉淀的样品相比，这些条带显示出更强的表达。接下来，我们尝试通过二维电泳溶解它们，并挑选候选点进行质谱分析。但这些蛋白质的含量很少，目前还不能得到可靠的结果。它无法在癌细胞中扩增这些蛋白质，因为我们还没有鉴定出来。现在我们试图增强HIFα与这些蛋白质之间的亲和力。我们改变培养条件，如氧气和/或葡萄糖浓度，以优化每个HIFα的表达，分别和检查在这样的条件下的亲和力。