Role of inactivation of components of mitochondrial complex II in the formation of paragangliomas/pheochromcytomas

编码线粒体复合物II亚基B、C和D（SDHB、SDHC、SDHD）的基因突变可引起副神经节瘤和嗜铬细胞瘤。SDHB、SDHC和SDHD似乎具有肿瘤抑制基因的功能。生殖系突变加上靶组织（神经嵴衍生组织）中野生型等位基因的缺失导致肿瘤形成。这种机制在SDHC和SDHD中得到了明确的证明，而在SDHB突变中则不那么令人信服。本申请旨在阐明在不存在SDHB、C或D的情况下导致肿瘤形成的分子机制。在PC 12和NIH/3 T3细胞中，使用基于质粒的系统，通过小干扰RNA（siRNA）使相应基因失活。将通过软琼脂试验研究细胞的集落形成，并通过电子显微镜研究超微结构形态，特别是线粒体。将测试呼吸链酶以研究敲低是否导致复合物II酶活性的损失。基因的表达模式将通过微阵列和实时定量PCR分析进行研究。特别强调将被放置在缺氧诱导因子1a和那些参与细胞周期的基因调控。

Mutations in genes coding for subunits B,C, and D of mitochondrial complex II (SDHB, SDHC, SDHD) can cause paragangliomas and pheochromocytomas. SDHB, SDHC, and SDHD appear to function as tumor suppressor genes. A germ line mutation plus a deletion of the wild-type allele in target tissues (neural-crest-derived tissues) result in tumor formation. This mechanism was clearly demonstrated for SDHC and SDHD, and somewhat less convincingly für SDHB mutations. This application aims at the elucidation of molecular mechanisms resulting in tumor formation in the absence of either SDHB, C, or D. The respective genes will be inactivated in both PC12 and NIH/3T3 cells by small interfering RNAs (siRNA) using a plasmid-based system. The cells will be investigated for colony formation by soft agar assays and the ultrastructural morphology, in paricular that of the mitochondria, will be studied by electron microscopy. Respiratory chain enzymes will be tested to investigate wheter the knock-downs resulted in loss of complex II enzymatic activity. Expression pattern of genes will be investigated by microarray and realtime quantitative PCR analyses. Special emphasis will be placed on genes regulated by hypoxia inducible factor 1a and those involved in the cell cycle.