Radiosensitization of noradrenaline transporter-expressing tumour cells by proteasome inhibitors and the role of reactive oxygen species

Background The radiopharmaceutical 131I-metaiodobenzylguanidine (131I-MIBG) is used for the targeted radiotherapy of noradrenaline transporter (NAT)-expressing neuroblastoma. Enhancement of 131I-MIBG's efficacy is achieved by combination with the topoisomerase I inhibitor topotecan - currently being evaluated clinically. Proteasome activity affords resistance of tumour cells to radiation and topoisomerase inhibitors. Therefore, the proteasome inhibitor bortezomib was evaluated with respect to its cytotoxic potency as a single agent and in combination with 131I-MIBG and topotecan. Since elevated levels of reactive oxygen species (ROS) are induced by bortezomib, the role of ROS in tumour cell kill was determined following treatment with bortezomib or the alternative proteasome inhibitor, MG132.<p></p> Methods Clonogenic assay and growth of tumour xenografts were used to investigate the effects of proteasome inhibitors alone or in combination with radiation treatment. Synergistic interactions in vitro were evaluated by combination index analysis. The dependency of proteasome inhibitor-induced clonogenic kill on ROS generation was assessed using antioxidants.<p></p> Results Bortezomib, in the dose range 1 to 30 nM, decreased clonogenic survival of both SK-N-BE(2c) and UVW/NAT cells, and this was prevented by antioxidants. It also acted as a sensitizer in vitro when administered with X-radiation, with 131I-MIBG, or with 131I-MIBG and topotecan. Moreover, bortezomib enhanced the delay of the growth of human tumour xenografts in athymic mice when administered in combination with 131I-MIBG and topotecan. MG132 and bortezomib had similar radiosensitizing potency, but only bortezomib-induced cytotoxicity was ROS-dependent.<p></p> Conclusions Proteasome inhibition shows promise for the treatment of neuroblastoma in combination with 131I-MIBG and topotecan. Since the cytotoxicity of MG132, unlike that of bortezomib, was not ROS-dependent, the latter proteasome inhibitor may have a favourable toxicity profile in normal tissues.<p></p&gt

DiffCoEx: a simple and sensitive method to find differentially coexpressed gene modules

Background: Large microarray datasets have enabled gene regulation to be studied through coexpression analysis. While numerous methods have been developed for identifying differentially expressed genes between two conditions, the field of differential coexpression analysis is still relatively new. More specifically, there is so far no sensitive and untargeted method to identify gene modules (also known as gene sets or clusters) that are differentially coexpressed between two conditions. Here, sensitive and untargeted means that the method should be able to construct de novo modules by grouping genes based on shared, but subtle, differential correlation patterns. Results: We present DiffCoEx, a novel method for identifying correlation pattern changes, which builds on the commonly used Weighted Gene Coexpression Network Analysis (WGCNA) framework for coexpression analysis. We demonstrate its usefulness by identifying biologically relevant, differentially coexpressed modules in a rat cancer dataset. Conclusions: DiffCoEx is a simple and sensitive method to identify gene coexpression differences between multiple conditions

Immunosuppressive role of fibrinogen-like protein 2 (FGL2) in CD8+regulatory T cells-mediated long-term graft survival

International audienc

ZyFISH: A Simple, Rapid and Reliable Zygosity Assay for Transgenic Mice

Microinjection of DNA constructs into fertilized mouse oocytes typically results in random transgene integration at a single genomic locus. The resulting transgenic founders can be used to establish hemizygous transgenic mouse lines. However, practical and experimental reasons often require that such lines be bred to homozygosity. Transgene zygosity can be determined by progeny testing assays which are expensive and time-consuming, by quantitative Southern blotting which is labor-intensive, or by quantitative PCR (qPCR) which requires transgene-specific design. Here, we describe a zygosity assessment procedure based on fluorescent in situ hybridization (zyFISH). The zyFISH protocol entails the detection of transgenic loci by FISH and the concomitant assignment of homozygosity using a concise and unbiased scoring system. The method requires small volumes of blood, is scalable to at least 40 determinations per assay, and produces results entirely consistent with the progeny testing assay. This combination of reliability, simplicity and cost-effectiveness makes zyFISH a method of choice for transgenic mouse zygosity determinations

Genetical genomics: spotlight on QTL hotspots

No abstract available

Expression quantitative trait loci are highly sensitive to cellular differentiation state

Blood cell development from multipotent hematopoietic stem cells to specialized blood cells is accompanied by drastic changes in gene expression for which the triggers remain mostly unknown. Genetical genomics is an approach linking natural genetic variation to gene expression variation, thereby allowing the identification of genomic loci containing gene expression modulators (eQTLs). In this paper, we used a genetical genomics approach to analyze gene expression across four developmentally close blood cell types collected from a large number of genetically different but related mouse strains. We found that, while a significant number of eQTLs (365) had a consistent “static” regulatory effect on gene expression, an even larger number were found to be very sensitive to cell stage. As many as 1,283 eQTLs exhibited a “dynamic” behavior across cell types. By looking more closely at these dynamic eQTLs, we show that the sensitivity of eQTLs to cell stage is largely associated with gene expression changes in target genes. These results stress the importance of studying gene expression variation in well-defined cell populations. Only such studies will be able to reveal the important differences in gene regulation between different ce

Reasoning with the HERMIT: tool support for equational reasoning on GHC core programs

A benefit of pure functional programming is that it encourages equational reasoning. However, the Haskell language has lacked direct tool support for such reasoning. Consequently, reasoning about Haskell programs is either performed manually, or in another language that does provide tool support (e.g. Agda or Coq). HERMIT is a Haskell-specific toolkit designed to support equational reasoning and user-guided program transformation, and to do so as part of the GHC compilation pipeline. This paper describes HERMIT’s recently developed support for equational reasoning, and presents two case studies of HERMIT usage: checking that type-class laws hold for specific instance declarations, and mechanising textbook equational reasoning

Deficiency and Also Transgenic Overexpression of Timp-3 Both Lead to Compromised Bone Mass and Architecture In Vivo

Tissue inhibitor of metalloproteinases-3 (TIMP-3) regulates extracellular matrix via its inhibition of matrix metalloproteinases and membrane-bound sheddases. Timp-3 is expressed at multiple sites of extensive tissue remodelling. This extends to bone where its role, however, remains largely unresolved. In this study, we have used Micro-CT to assess bone mass and architecture, histological and histochemical evaluation to characterise the skeletal phenotype of Timp-3 KO mice and have complemented this by also examining similar indices in mice harbouring a Timp-3 transgene driven via a Col-2a-driven promoter to specifically target overexpression to chondrocytes. Our data show that Timp-3 deficiency compromises tibial bone mass and structure in both cortical and trabecular compartments, with corresponding increases in osteoclasts. Transgenic overexpression also generates defects in tibial structure predominantly in the cortical bone along the entire shaft without significant increases in osteoclasts. These alterations in cortical mass significantly compromise predicted tibial load-bearing resistance to torsion in both genotypes. Neither Timp-3 KO nor transgenic mouse growth plates are significantly affected. The impact of Timp-3 deficiency and of transgenic overexpression extends to produce modification in craniofacial bones of both endochondral and intramembranous origins. These data indicate that the levels of Timp-3 are crucial in the attainment of functionally-appropriate bone mass and architecture and that this arises from chondrogenic and osteogenic lineages

XGAP: a uniform and extensible data model and software platform for genotype and phenotype experiments.

We present an extensible software model for the genotype and phenotype community, XGAP. Readers can download a standard XGAP (http://www.xgap.org) or auto-generate a custom version using MOLGENIS with programming interfaces to R-software and web-services or user interfaces for biologists. XGAP has simple load formats for any type of genotype, epigenotype, transcript, protein, metabolite or other phenotype data. Current functionality includes tools ranging from eQTL analysis in mouse to genome-wide association studies in humans.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector

BACKGROUND: Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers. METHODS: We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codon-swapped ZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn rats were used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors to deliver ZFNs into target cells. RESULTS: We reduced total identity between ZFN monomers from 90.9% to 61.4% and showed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codon-swapping, leading to much higher ZFN activity in cell lines (up to 7-fold increase compared to unmodified ZFNs and 60% activity in C6 cells), as compared to plasmid transfection or a single ICLV encoding unmodified ZFN monomers. Off-target analysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first time successful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after delivery of a single IDLV encoding two ZFNs. CONCLUSION: These results demonstrate that a codon-swapping approach allowed a single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFN-mediated genome editing of primary cells, for both ex vivo or in vivo applications