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GADD45a as a biomarker of DNA damage : investigations into the specificity of the GreenScreen HC genotoxicity assayTopham, Caroline January 2010 (has links)
Recent studies have highlighted the poor specificity of a battery of in vitro genotoxicity tests when predicting rodent carcinogenicity, in some cases leading to misleading predictions of genotoxic carcinogenicity. GreenScreen HC (GSHC) is a highly sensitive human cell-based genotoxicity assay that, in contrast to the battery in vitro mammalian cell tests, also exhibits high specificity. GSHC employs the response of the human DNA damage-inducible gene GADD45a as a marker of genotoxic stress, using a GFP reporter construct hosted by the TK6 cell line. In order to better understand the biological basis for the high specificity of GSHC, three approaches were taken. Firstly, the relevance of the choice of a p53-competent host cell line, TK6, was investigated. A database was compiled consisting of published genotoxicity data from the in vitro battery tests performed in TK6 cells, and comparative GSHC data generated. This work revealed that discordance existed between the tests analysed, therefore the specificity of GSHC was not dictated by the choice of cell line alone. To confirm that misleading positive results could still be generated in the GSHC TK6 cell line, mutation assays were performed for compounds with pre-existing misleading positive battery test data, and a dose-dependent increase in mutation frequency was observed for 1 of 3 compounds. Secondly, the importance of wild-type p53 to the specificity of GSHC was investigated by generating p53-deficient GADD45a-GFP reporter cell lines. This work identified wild-type p53 as a key regulator of GADD45a in the context of GSHC, with reduced expression levels observed in response to genotoxic stimuli. Finally NF-kB, a putative direct regulator of GADD45a in response to DNA damage, was shown to contribute to the expression of GADD45a at high doses of 4-nitroquinoline-N-oxide via mutational analysis of two NF-kB binding sites in the promoter and intron 3 of the GADD45a-GFP reporter. This implicates NF-kB in the direct regulation of GADD45a in response to genotoxic stress in the context of GSHC. The work presented here demonstrates that the high specificity of GSHC is not dictated solely by the use of the p53-competent TK6 cell line, but is due to the use of a unique biosensor of genotoxic stress; GADD45a expression. Interference with the function of two key stress response signalling nodes, p53 and NF-kB, revealed that these regulators dictate the magnitude of expression of GADD45a, directly impacting upon the specificity of GSHC.
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REGULATION OF STRESS-ACTIVATED MAP KINASE PATHWAYS DURING CELL FATE DECISIONSICHIKAWA, KENJI, NAKAMURA, TAKANORI, KUBOTA, YUJI, TAKEKAWA, MUTSUHIRO 02 1900 (has links)
No description available.
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Environmental and genetic modifiers of Shudderer, a Drosophila voltage-gated sodium channel mutantChen, Hung-Lin 01 August 2016 (has links)
There is a complex relationship between genetic mutations and their phenotypic expression. Two patients who carry the exactly same disease-causing mutation can have drastically different severity in disease symptoms. Such phenotypic variations may be due to environmental factors, such as diet, stress and temperature, as well as genetic variations, including single nucleotide polymorphisms and copy number variations in other loci. From a clinical point of view, the environmental and genetic factors that modify phenotypic severity are important because, even when we cannot correct the original mutations, it may be able to reduce the burden of genetically inherited disorders by manipulating these modifiers. To study environmental and genetic modifiers, we used Shudderer (Shu), a Drosophila mutant for the voltage-gated sodium (Nav) channel gene, as an experimental model. Nav channels are essential for generation and propagation of action potentials in neurons. Reflecting their functional importance in neural function, mutations in the Nav channel genes are associated with a variety of human neurological disorders. Shu mutants display severe behavioral defects (e.g., spontaneous jerking and heat-induced seizures) and morphological abnormalities (e.g., indented thorax and down-turned wings). The goal of this study was to identify and characterize the environmental and genetic factors that modify behavioral and morphological phenotypes of Shu mutants. For environmental modifiers, we serendipitously discovered that a diet supplemented with milk dramatically reduces the Shu phenotypes. To identify genetic modifiers, we took two independent approaches, microarray analysis and unbiased forward genetic modifier screening. We found that reduction of Gadd45 or GstS1 activity leads to suppression of the Shu phenotypes to different degrees. Intriguingly, the effects of these genetic and environmental modifiers apparently converge into enhancement of the GABAergic inhibitory system. Because the molecular and cellular mechanisms underlying the basic neurobiological processes are highly conserved between flies and humans, our findings are expected to provide fundamental insights into genetic and environmental modifiers for human Nav channel gene mutations, leading to the future development of novel strategies for preventing and treating disorders caused by dysfunctional Navchannels.
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Molecular Mechanism of Vitamin D Action and its Implications in Ovarian Cancer Prevention and TherapyJiang, Feng 01 May 2004 (has links)
1,25-dihydroxyvitamin D3 (1,25VD), the active form of vitamin D (VD), suppresses the growth of numerous human cancer cell lines by inhibiting cell cycle progression and inducing cell death. Genes that mediate each of these activities remain largely unidentified and there are no preclinical data for 1,25VD analogues in ovarian cancer (OCa). We hypothesize that 1,25VD and its analogues inhibit the development of OCa. In this study, we demonstrated, (a) 1,25VD causes cell cycle arrest at the G1/S and G2/M transition and induces apoptosis in OCa cells. (b) We also found that gadd45 is one of primary target genes for 1,25VD-mediated G2/M arrest. A direct repeat 3 (DR3) vitamin D response element (VDRE) is identified in the fourth exon of gadd45. This exonic VDRE forms a complex with the vitamin D receptor (VDR)/retinoid X receptor (RXR) heterodimer in vitro and mediates the induction of reporter activity by 1,25VD in vivo. VDR is recruited in a ligand-dependent manner to the exonic enhancer but not to the gadd45 promoter regions. In OCa cells expressing GADD45 anti-sense cDNA or GADD45-null mouse embryo fibroblasts, 1,25VD fails to induce G2/M arrest, suggesting that G2/M arrest induced by 1,25VD is mediated through GADD45. Further study showed that GADD45 mediates the effect of 1,25VD by decreasing cdc2 kinase activity. (c) hTERT, the catalytic subunit of telomerase, is identified as a primary target for 1,25VD. 1,25VD decreases telomerase activity and hTERT mRNA expression. The down-regulation of hTERT mRNA is due to decreased mRNA stability by 1,25VD, rather than decreased transcription of hTERT through VDRE. Clones stably transfected with hTERT showed higher telomerase activity and longer telomere length than parental cells. Moreover, hTERT clones resist 1,25VD-induced apoptosis and growth inhibition. In contrast to parental cells which do not recover from prolonged treatment with 1,25VD, hTERT clones re-grew rapidly after 1,25VD withdrawal. (d) We demonstrated that the 1,25VD analogue EB1089 inhibits OCa cells in vitro and OCa xenograft in vivo without inducing hypercalcemia. We also demonstrated precursors for epithelial OCa express VDR and human primary ovarian surface epithelial cells respond to 1,25VD. Taken together, these results strongly suggest that 1,25VD analogues may be effective in the chemoprevention and chemotherapy of OCa.
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