Publications (48) View all
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Article: A novel mechanism of sodium iodide symporter repression in differentiated thyroid cancer.
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ABSTRACT: Differentiated thyroid cancers and their metastases frequently exhibit reduced iodide uptake, impacting on the efficacy of radioiodine ablation therapy. PTTG binding factor (PBF) is a proto-oncogene implicated in the pathogenesis of thyroid cancer. We recently reported that PBF inhibits iodide uptake, and have now elucidated a mechanism by which PBF directly modulates sodium iodide symporter (NIS) activity in vitro. In subcellular localisation studies, PBF overexpression resulted in the redistribution of NIS from the plasma membrane into intracellular vesicles, where it colocalised with the tetraspanin CD63. Cell-surface biotinylation assays confirmed a reduction in plasma membrane NIS expression following PBF transfection compared with vector-only treatment. Coimmunoprecipitation and GST-pull-down experiments demonstrated a direct interaction between NIS and PBF, the functional consequence of which was assessed using iodide-uptake studies in rat thyroid FRTL-5 cells. PBF repressed iodide uptake, whereas three deletion mutants, which did not localise within intracellular vesicles, lost the ability to inhibit NIS activity. In summary, we present an entirely novel mechanism by which the proto-oncogene PBF binds NIS and alters its subcellular localisation, thereby regulating its ability to uptake iodide. Given that PBF is overexpressed in thyroid cancer, these findings have profound implications for thyroid cancer ablation using radioiodine.Journal of Cell Science 09/2009; 122(Pt 18):3393-402. · 6.11 Impact Factor -
Article: Profiling RNA interference (RNAi)-mediated toxicity in neural cultures for effective short interfering RNA design.
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ABSTRACT: A promising strategy to enhance axon regeneration is to employ short interfering (si)RNA targeting either RhoA or p75(NTR), which are components of a signalling cascade triggered by growth inhibitory ligands. However, it is important to profile the biological impact of siRNA on cell homeostasis in order to develop safe and effective therapies. We used microarray and quantitative reverse transcriptase-polymerase chain reaction techniques to analyse the transcriptional effects of siRNA against p75(NTR) and RhoA in neuronal cell line and primary cultures. Expression analysis showed that primary rat dorsal root ganglion cells were up to 279-fold more sensitive than nerve growth factor-differentiated PC12 cells in detecting innate immune responses to siRNA. The sequence and method of synthesis of siRNA critically influenced the magnitude of the innate immune response. Importantly, siRNA sequences were identified that efficiently silenced RhoA and p75(NTR) mRNA with attenuated induction of the interferon-responsive gene mx1. Moreover, microarray analysis identified genes related to RhoA function, such as tgf beta 2, plod2 and mmp3, with implications for interpretating the ability of RhoA siRNA to promote axon regeneration. These findings demonstrate the importance of screening the biological impact of different siRNA sequences not only for their silencing efficacy, but also for potential toxicity. The results of the present study suggest that the toxicity observed was sequence-dependent because only two out of five siRNA sequences targeting RhoA were identified that did not induce a significant innate immune response.The Journal of Gene Medicine 04/2009; 11(6):523-34. · 2.48 Impact Factor -
Article: Optimisation of siRNA-mediated RhoA silencing in neuronal cultures
Molecular and Cellular Neuroscience 04/2009; 40(4):451. · 3.66 Impact Factor -
Article: In vitro evaluation of a 'stealth' adenoviral vector for targeted gene delivery to adult mammalian neurones.
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ABSTRACT: Polymer coating of adenovirus type 5 (Ad5) particles produces a 'stealth' Ad5 (sAd5) that confers protection from immune recognition, blocks receptor-mediated uptake, and favours uptake into pinocytic cells. In mixed cultures of primary adult rat dorsal root ganglion neurones (DRGN), rat C6 glioma cells, A9 non-Coxsackie and Ad Receptor (CAR)- and CAR-expressing fibroblasts, reporter gene expression after sAd5 pinocytotic uptake was monitored using the green fluorescent protein (gfp) gene, and viral particle trafficking and polymer coat dismantling was followed using Yoyo-1 tagged Ad5 DNA and Texas Red (TR) to label the coat. sAd5.gfp was pinocytosed by significantly higher proportions of neurones, than other cells, but GFP was not expressed. The TR-labelled coat remained co-localised with tagged viral DNA within transfected DRGN, showing that sAd5 did not uncoat and viral DNA did not traffic to the nucleus. Noncoated Ad5 transduced non-neuronal DRG cells more efficiently than DRGN, whereas A9(CAR) cells were more significantly transduced than any other cell type. Retargeting of the sAd5.gfp with either fibroblast growth factor-2 or nerve growth factor (NGF) enhanced internalisation by DRGN into endocytic vesicles allowing uncoating and thus GFP expression. Retargeting with NGF resulted in significantly higher numbers of DRGN expressing GFP than non-neuronal DRG cells. These findings indicate that DRGN pinocytose atropic genetic particles at higher levels than non-neuronal DRG cells and the environment of pinocytic vesicles is not conducive to sAd5 uncoating and capsid dismantling, requiring reformulation of sAd5 with either a neurone specific ligand or a self-dismantling coat to target sAd5 transgene expression to neurones.The Journal of Gene Medicine 02/2009; 11(4):335-44. · 2.48 Impact Factor -
Article: Targeting adenoviral transgene expression to neurons.
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ABSTRACT: Adenovirus (Ad) is an efficient and safe vector for CNS gene delivery since it infects non-replicating neurons and does not cause insertional mutagenesis of host cell genomes. However, the promiscuous Ad CAR receptor targets cells non-specifically and activates a host immune response. Using Ad5 containing an expression cassette encoding the gene for green fluorescent protein, gfp, regulated by the neuron specific promoter synapsin-1 and the woodchuck post-transcriptional regulatory element (WPRE), we demonstrate efficient, prolonged and promoter-restricted gfp expression in neurons of mixed primary adult rat dorsal root ganglion (DRG) and retinal cell cultures. We also demonstrate restricted gfp expression in DRG neurons after direct injections of Ad5 containing the synapsin-1(gfp)/WPRE construct into L4 DRG in vivo, while Ad5 CMV(gfp) transfected both DRG glia and neurons. Moreover, since the effective titres of delivered Ad5 are reduced with this neuron specific promoter/WPRE expression cassette, the viral immune challenge should be attenuated when used in vivo.Molecular and Cellular Neuroscience 09/2008; 39(3):411-7. · 3.66 Impact Factor
