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

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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.