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

(导言)肾细胞癌标本表现出不同的缺氧诱导因子1α和2α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。为了阐明这一点，我们寻找每个HIFα的特异结合蛋白。(材料与方法)从肾癌细胞株ACHN和SN12C中提取在低氧条件下具有正常von Hippel-Lindau(VHL)蛋白功能和表达两种HIFα的蛋白，免疫沉淀寻找与HIFα特异性抗体结合的蛋白。用SDS-PAGE对免疫共沉淀蛋白进行溶解，并用银染法比较表达水平。(结果与未来计划)我们可以检测到几条候选条带，与对照抗体共沉淀的样品相比，表达更强。下一步，我们尝试用双向电泳法将它们溶解，并挑选候选点进行质谱分析。但这些蛋白质的量很少，到目前为止，我们还没有得到可靠的结果。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，我们试图提高缺氧诱导因子αS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，分别优化每个缺氧诱导因子α的表达，并在这样的条件下检查亲和力。

(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.