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

线粒体复合体II(SDHB、SDHC、SDHD)B、C和D亚单位编码基因的突变可导致副神经节瘤和嗜铬细胞瘤。SDHB、SDHC和SDHD似乎是肿瘤抑制基因。生殖系突变加上目标组织(神经脊源性组织)中野生型等位基因的缺失会导致肿瘤的形成。这一机制在SDHC和SDHD中得到了明确的证明，而在für SDHB突变中则不那么令人信服。这项应用旨在阐明在缺乏SDHB、C或D的情况下导致肿瘤形成的分子机制。在PC12和NIH/3T3细胞中，各自的基因将通过小干扰RNA(SiRNA)使用基于质粒的系统来失活。细胞的集落形成将通过软琼脂试验进行研究，超微结构的形态，尤其是线粒体的超微结构，将通过电子显微镜进行研究。呼吸链酶将进行测试，以调查击倒是否导致复合体II酶活性的丧失。基因的表达模式将通过基因芯片和实时定量聚合酶链式反应进行研究。将特别强调受低氧诱导因子1a调节的基因和那些参与细胞周期的基因。

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.