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

（导读）肾细胞癌标本显示缺氧诱导因子（HIF） 1α和2α的不同染色模式。HIF1α和2α同源性高，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。为了澄清这一点，我们为每个HIFα寻找特定的结合蛋白。（材料与方法）从肾细胞癌细胞株ACHN和SN12C中提取VHL蛋白功能正常且在缺氧条件下表达HIFαs的蛋白，并进行免疫沉淀，寻找与HIFα特异性抗体结合的特异性蛋白。SDS-PAGE溶解共免疫沉淀蛋白，银染色比较表达水平。（结果及未来计划）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，这些条带的表达更强。接下来，我们尝试用2d电泳溶解它们，并挑选候选点进行质谱分析。但是这些蛋白质的数量太少了，到目前为止我们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，因为我们还没有确定。现在我们试图增强HIFαs与这些蛋白之间的亲和力。我们改变培养条件，如氧气和/或葡萄糖浓度，分别优化每个HIFα表达，并检查在这种条件下的亲和力。

(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.