DECIPHERING THE MECHANISMS OF MEDULLOBLASTOMA RESISTANCE TO RADIO/CHEMOTHERAPY: TOWARD PERSONALIZED TREATMENTS?

International audienc

Identification of a Variable Number of Tandem Repeats Polymorphism and Characterization of LEF-1 Response Elements in the Promoter of the IDO1 Gene

Indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step of the kynurenine pathway that is an important component of immunomodulatory and neuromodulatory processes. The IDO1 gene is highly inducible by IFN-γ and TNF-α through interaction with cis-acting regulatory elements of the promoter region. Accordingly, functional polymorphisms in the IDO1 promoter could partly explain the interindividual variability in IDO expression that has been previously documented.A PCR-sequencing strategy, applied to DNA samples from healthy Caucasians, allowed us to identify a VNTR polymorphism in the IDO1 promoter, which correlates significantly with serum tryptophan concentration, controlled partially by IDO activity, in female subjects, but not in males. Although this VNTR does not appear to affect basal or cytokine-induced promoter activity in gene reporter assays, it contains novel cis-acting elements. Three putative LEF-1 binding sites, one being located within the VNTR repeat motif, were predicted in silico and confirmed by chromatin immunoprecipitation. Overexpression of LEF-1 in luciferase assays confirmed an interaction between LEF-1 and the predicted transcription factor binding sites, and modification of the LEF-1 core sequence within the VNTR repeat motif, by site-directed mutagenesis, resulted in an increase in promoter activity.The identification of a VNTR in the IDO1 promoter revealed a cis-acting element interacting with the most downstream factor of the Wnt signaling pathway, suggesting novel mechanisms of regulation of IDO1 expression. These data offer new insights, and suggest further studies, into the role of IDO in various pathological conditions, particularly in cancer where IDO and the Wnt pathway are strongly dysregulated

Induction of TDO2 and IDO2 in Liver by High-Fat Feeding in Mice: Discrepancies with Human Obesity

Low-grade and chronic inflammation is elicited in white adipose tissue in human obesity. The presence of inflammatory molecules leads to an increased tryptophan catabolism through the induction of indoleamine-2,3-dioxygenase-1 (IDO1). In order to characterize the mechanisms underlying this dysregulation, we have studied 2 mouse models of obesity. Unexpectedly, we did not detect any IDO1 expression in obese or lean mice adipose tissue. In a previous study, we did not find any significant difference in the liver for IDO2 and tryptophan-2,3-dioxygenase (TDO2) gene expression between normal weight and obese patients. IDO2 and TDO2 expression was increased in the liver of high-fat fed mice, but not in ob/ob mice, and was strongly correlated with hydroxysteroid-(11-beta) dehydrogenase-1 (HSD11B1) expression, an enzyme that generates active cortisol within tissues. In conclusion, despite a dysregulation of tryptophan metabolism, obese mice display discrepancies with human obesity metabolism, rendering them inappropriate for further investigations in this animal model

Improved donor/acceptor BRET couples for monitoring ß-arrestin recruitment to G protein-coupled receptors

International audienceWe report highly sensitive Bioluminescence Resonance Energy Transfer (BRET) assays with optimized donor/acceptor couples. We combined the energy donors Renilla luciferase (Rluc) and the Rluc8 variant with the energy acceptors yellow fluorescent protein (YFP), the YPet variant and the Renilla green fluorescent protein (RGFP). Different donor/acceptor couples were tested in well-established assays measuring ligand-induced ß-arrestin (ßARR) intramolecular rearrangements and recruitment to G protein-coupled receptors (GPCRs). We show increased sensitivity with Rluc8/YPet and Rluc8/RGFP couples and measured previously undetectable BRET signals. These tools improve existing ßARR assays and offer new options for the development of future BRET assays

Low-dose exposure to bisphenols A, F and S of human primary adipocyte impacts coding and non-coding RNA profiles.

Bisphenol A (BPA) exposure has been suspected to be associated with deleterious effects on health including obesity and metabolically-linked diseases. Although bisphenols F (BPF) and S (BPS) are BPA structural analogs commonly used in many marketed products as a replacement for BPA, only sparse toxicological data are available yet. Our objective was to comprehensively characterize bisphenols gene targets in a human primary adipocyte model, in order to determine whether they may induce cellular dysfunction, using chronic exposure at two concentrations: a "low-dose" similar to the dose usually encountered in human biological fluids and a higher dose. Therefore, BPA, BPF and BPS have been added at 10 nM or 10 μM during the differentiation of human primary adipocytes from subcutaneous fat of three non-diabetic Caucasian female patients. Gene expression (mRNA/lncRNA) arrays and microRNA arrays, have been used to assess coding and non-coding RNA changes. We detected significantly deregulated mRNA/lncRNA and miRNA at low and high doses. Enrichment in "cancer" and "organismal injury and abnormalities" related pathways was found in response to the three products. Some long intergenic non-coding RNAs and small nucleolar RNAs were differentially expressed suggesting that bisphenols may also activate multiple cellular processes and epigenetic modifications. The analysis of upstream regulators of deregulated genes highlighted hormones or hormone-like chemicals suggesting that BPS and BPF can be suspected to interfere, just like BPA, with hormonal regulation and have to be considered as endocrine disruptors. All these results suggest that as BPA, its substitutes BPS and BPF should be used with the same restrictions

Number of differentially down and up-regulated miRNA probes amongst the 483 probes after the exposition of the pre-adipocytes to 10 nM or 10 μM of bisphenol (A, F and S) during differentiation shared between BPA and BPS or BPF.

<p>Number of differentially down and up-regulated miRNA probes amongst the 483 probes after the exposition of the pre-adipocytes to 10 nM or 10 μM of bisphenol (A, F and S) during differentiation shared between BPA and BPS or BPF.</p

Numbers of differentially down and up-regulated miRNA probes amongst the 483 probes after the exposition of the pre-adipocytes to 10 nM or 10 μM of bisphenol shared between the concentrations for each bisphenol.

<p>Numbers of differentially down and up-regulated miRNA probes amongst the 483 probes after the exposition of the pre-adipocytes to 10 nM or 10 μM of bisphenol shared between the concentrations for each bisphenol.</p

Enrichment analysis using Ingenuity Pathway Analysis on the lists of mRNA probes differentially expressed after exposition to the three bisphenols A, F and S at 10 nM and 10 μM.

<p>Enrichment analysis using Ingenuity Pathway Analysis on the lists of mRNA probes differentially expressed after exposition to the three bisphenols A, F and S at 10 nM and 10 μM.</p

List of differentially expressed probes deregulated for the three bisphenols A, F and S at 10 nM.

<p>List of differentially expressed probes deregulated for the three bisphenols A, F and S at 10 nM.</p

Heatmap representation of significantly differentially miRNA in the three bisphenols at 10 nM and 10 μM.

<p>The represented values are log₂ fold changes of the ratio of the expression in a given bisphenol and concentration over the control condition which is differentiated without any bisphenol.</p