2011 Fiscal Year Final Research Report

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

Research Project

Project/Area Number	21592054
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Urology
Research Institution	Yokohama City University
Principal Investigator	KONDO Keiichi 横浜市立大学, 市民総合医療センター, 准教授 (80363836)
Project Period (FY)	2009 – 2011
Keywords	HIF / 結合蛋白 / 低酸素環境 / 腎細胞癌 / 治療感受性
Research Abstract	(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.