5′HS5 of the Human β-globin Locus Control Region Is Dispensable for the Formation of the β-globin Active Chromatin Hub

Hypersensitive site 5 (5′HS5) of the β-globin Locus Control Region functions as a developmental stage-specific border in erythroid cells. Here, we have analyzed the role of 5′HS5 in the three dimensional organization of the β-gene locus using the Chromatin Conformation Capture (3C) technique. The results show that when 5′HS5 is deleted from the locus, both remote and internal regulatory elements are still able to interact with each other in a three-dimensional configuration termed the Active Chromatin Hub. Thus, the absence of 5′HS5 does not have an appreciable effect on the three dimensional organization of the β-globin locus. This rules out models in which 5′HS5 nucleates interactions with remote and/or internal regulatory elements. We also determined the binding of CTCF, the only defined insulator protein in mammalian cells, to 5′HS5 by using chromatin immunoprecipitation (ChIP) assays. We detect low levels of CTCF binding to 5′HS5 in primitive erythroid cells, in which it functions as a border element. Surprisingly, we also observe binding levels of CTCF to 5′HS5 in definitive erythroid cells. Thus, binding of CTCF to 5′HS5 per se does not render it a functional border element. This is consistent with the previous data suggesting that CTCF has dual functionality

The novel thiosemicarbazone, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), inhibits neuroblastoma growth in vitro and in vivo via multiple mechanisms

Abstract Background Neuroblastoma is a relatively common and highly belligerent childhood tumor with poor prognosis by current therapeutic approaches. A novel anti-cancer agent of the di-2-pyridylketone thiosemicarbazone series, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), demonstrates promising anti-tumor activity. Recently, a second-generation analogue, namely di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), has entered multi-center clinical trials for the treatment of advanced and resistant tumors. The current aim was to examine if these novel agents were effective against aggressive neuroblastoma in vitro and in vivo and to assess their mechanism of action. Methods Neuroblastoma cancer cells as well as immortalized normal cells were used to assess the efficacy and selectivity of DpC in vitro. An orthotopic SK-N-LP/Luciferase xenograft model was used in nude mice to assess the efficacy of DpC in vivo. Apoptosis in tumors was confirmed by Annexin V/PI flow cytometry and H&E staining. Results DpC demonstrated more potent cytotoxicity than Dp44mT against neuroblastoma cells in a dose- and time-dependent manner. DpC significantly increased levels of phosphorylated JNK, neuroglobin, cytoglobin, and cleaved caspase 3 and 9, while decreasing IkBα levels in vitro. The contribution of JNK, NF-ĸB, and caspase signaling/activity to the anti-tumor activity of DpC was verified by selective inhibitors of these pathways. After 3 weeks of treatment, tumor growth in mice was significantly (p < 0.05) reduced by DpC (4 mg/kg/day) given intravenously and the agent was well tolerated. Xenograft tissues showed significantly higher expression of neuroglobin, cytoglobin, caspase 3, and tumor necrosis factor-α (TNFα) levels and a slight decrease in interleukin-10 (IL-10). Conclusions DpC was found to be highly potent against neuroblastoma, demonstrating its potential as a novel therapeutic for this disease. The ability of DpC to increase TNFα in tumors could also promote the endogenous immune response to mediate enhanced cancer cell apoptosis

Chinese B thalassaemia: DNA polymorphisms andspecific mutations

published_or_final_versionBiochemistryDoctoralDoctor of Philosoph