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Tetracycline-Inducible Cyr61 effect on LN229 glioma cells

ABSTRACT: Glioblastoma multiforme is the most common and aggressive form of brain cancer. The use of oncolytic HSV-1 (oHSV) to selectively target brain cancer cells leading to their lytic destruction has shown to be very promising in a preclinical setting, but is lacking efficacy in clinical trials. Cyr61, a secreted extracellular matrix protein which functions to promote angiogenesis, migration, proliferation and tumorigenesis, was found to be upregulated rapidly following oHSV infection. Here we show, using microarray analysis, that Cyr61 expression leads to the induction of several genes with type 1 interferon function. We show that Cyr61 mediated type 1 IFN induction is through its interaction with integrin alpha6beta1 on the cell surface and results in oHSV inhibition, reducing the efficacy of this therapy. We used microarray to detail the global program of gene expression underlying Cyr61 mediated oncolytic HSV-1 inhibition and identified distinct classes of up-regulated genes during this process. Tetracycline-Inducible glioma cells expressing Cyr61 protein in the presence of doxycycline were treated with or without doxycycline for 24 hours. RNA was extracted and hybridized on Affymetrix microarray. Two groups: ± dox to induce cyr61, performed in triplicate.

ORGANISM(S): Homo sapiens

SUBMITTER: Haseley A

PROVIDER: S-ECPF-GEOD-29384 | biostudies-other | 2012 Mar

REPOSITORIES: biostudies-other

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Project description:IntroductionThe controlled expression of many genes, including G-protein coupled receptors (GPCRs), is important for delineating gene functions in complex model systems. Binary systems for inducible regulation of transgene expression are widely used in mice. One system is the tTA/TRE expression system, composed of a tetracycline-dependent DNA binding factor and a separate tetracycline operon. However, the requirement for two separate transgenes (one for each tTA or TRE component) makes this system less amenable to models requiring directed cell targeting, increases the risk of multiple transgene integration sites, and requires extensive screening for appropriately-functioning clones.MethodsWe developed a single, polycistronic tetracycline-inducible expression platform to control the expression of multiple cistrons in mammalian cells. This platform has three basic constructs: regulator, responder, and destination vectors. The modular platform is compatible with both the TetOff (tTA) and TetOn (rtTA) systems. The modular Gateway recombineering-compatible components facilitate rapidly generating vectors to genetically modify mammalian cells. We apply this system to use the elongation factor 1? (EF1?) promoter to drive doxycycline-regulated expression of both the fluorescent marker mCherry and an engineered Gs-coupled GPCR "Rs1" separated by a 2A ribosomal skip site.ResultsWe show that our combined expression construct drives expression of both the mCherry and Rs1 transgenes in a doxycycline-dependent manner. We successfully target the expression construct into the Rosa26 locus of mouse embryonic stem (ES) cells. Rs1 expression in mouse ES cells increases cAMP accumulation via both basal and ligand-induced Gs mechanisms and is associated with increased embryoid body size. Heterozygous mice carrying the Rs1 expression construct showed normal growth and weight, and developed small increases in bone formation that could be observed in the calvaria.ConclusionsOur results demonstrate the feasibility of a single-vector strategy that combines both the tTA and TRE tetracycline-regulated components for use in cells and mouse models. Although the EF1? promoter is useful for driving expression in pluripotent cells, a single copy of the EF1? promoter did not drive high levels of mCherry and Rs1 expression in the differentiated tissues of adult mice. These findings indicate that promoter selection is an important factor when developing transgene expression models.

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Tetracycline-Inducible Cyr61 effect on LN229 glioma cells

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