Development of novel ill vitro and ill vivo models for determining primary events in HLRCC tumourigenesis Linda O'Flaherty, Mansfield College Thesis submitted for degree of Doctor of Philosophy Nuffield Department of Clinical Medicine, University of Oxford Hilary Term 2012 Germline mutations of fumarate hydratase (FR), encoding an enzyme of the tricarboxylic acid (TCA) cycle, predispose affected individuals to hereditary leiomyomatosis and renal cell cancer (HLRCC). FH-deficient cells and tissues have been shown to accumulate fumarate, exhibit S-(2-succinyl) cysteine (2SC) protein modifications and to constitutively express hypoxia-inducible factor alpha (HIF -1 a and -20.), under nonnoxic conditions. This thesis presents a phenotypic characterisation of FhI-I- mouse embryonic fibroblasts (MEFs), generated from previously reported conditional Fhl knockout mice, as a new in vitro system for investigating and identifying biochemical and metabolic pathways that are dysregulated as a result of FhI inactivation. These cell lines reproduced the aforementioned phenotypes, in addition to an observed shift from oxidative phosphorylation (OXPHOS) to glycolytic metabolism. Re-expression of either full length, mitochondrial-targeted FH (FhI-I- +FH) or cytoplasmic FH (Fhrl- +FHl'1MTS) in FhI-deficient MEFs was sufficient to reduce intracellular fumarate and to correct for the dysregulation of the Hif pathway. These results were of particular interest as they demonstrated that nonnoxic stabilisation of Hif-Ia occurs independently of the persistent mitochondrial defect observed in Fhrl- +FHl'1MTS MEFs. These findings were corroborated in vivo following the development of transgenic mouse models, ubiquitously expressing either FH or FHl'1MTS in mice with targeted inactivation of FhI in renal tubular cells. Surprisingly, the cytoplasmic-restricted FH (FHl'1MTS) transgene was just as efficient as the transgenic mice expressing mitochondrial- targeted FH at rescuing the cystic phenotype associated with Fh I-deficiency in the kidneys. As the function of cytoplasmic FH has remained poorly understood, these results go some way to extricating a role for this isofonn of FH. The results of this thesis demonstrate that these novel in vitro and in vivo models, used either alone or in combination, are a versatile and robust paradigm for studying altered cell metabolism in not only HLRCC but other diseases associated with metabolic dysregulation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:565984 |
| Date | January 2012 |
| Creators | O'Flaherty, Linda H. |
| Contributors | Ratcliffe, Peter ; Pollard, Patrick |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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