Caractérisation fonctionnelle d’ARN non codants eucaryotes par des approches de biologie moléculaire et de phylogénétique

Les petits ARN nucléolaires (snoARN) sont des ARN non codants qui très généralement guident des enzymes de modification des ARN par appariement de bases avec des cibles ARN ribosomiques (ARNr). Cependant, les données récentes de la bibliographie suggèrent que les snoARN peuvent également réaliser des fonctions non classiques qui semblent principalement affecter le destin d’autres classes d’ARN mais sont pour l’instant mal documentées. Le syndrome de Prader Willi (SPW) est une pathologie neurocomportementale rare pour laquelle il n’existe pas de traitement curatif ; une caractéristique des patients SPW est l’absence d’expression des snoARN Snord116, dont les fonctions sont très mal comprises. Nos travaux antérieurs au projet ont suggéré que les Snord116 s’apparient avec des ARNm cibles spécifiques et affectent leur niveau d’expression à l’équilibre et leur épissage. A partir de ces données, le premier axe du projet vise à caractériser les fonctions moléculaires des Snord116 afin d’améliorer la compréhension de leur mécanisme d’action et de développer une stratégie à visée thérapeutique de correction de la perte d’expression des Snord116 dans le SPW. Le second axe du projet cherchera à identifier de nouveaux snoARN fonctionnels en appliquant les méthodologies – notamment phylogénétiques– utilisées dans nos travaux antérieurs et, par suite, à caractériser leurs fonctions par des approches de biologie moléculaire. A terme, la pertinence du déploiement de ces approches vers d’autres catégories d’ARN non codants sera évaluée

Élément invMED1 et complexe LRP130 d'activation du gène MDR1 humain (découverte et application à la génothérapie du phénotype de multi-chimiorésistance des cancers)

Le phénotype de résistance des cancers à la chimiothérapie (MRD) est principalement dû à la surproduction de protéines membranaires telles que la Pgp codée par le gène MDR1 chez l'humain. Nous rapportons ici la découverte d'un élément cis-activateur de la transcription des gènes MDR1 et MVP impliqués dans le phénotype MDR, que nous avons nommé invMED1. Nous avons étudié un facteur nucléaire de 150 kDa dont l'intensité d'interaction avec l'élément invMED1 croît lorsque le niveau de chimiorésistance intrinsèque de cellules cancéreuses en culture augmente. La caractérisation de ce facteur a permis d'indentifier sa copmposante protéique, la protéine LRP130, et suggère la présence d'un partenaire ARN. Ces détails permettent de développer nos stratégies de génothérapie du phénotype MDR. Grâce à l'établissement de lignées cancéreuses issues de mélanomes uvéaux humains et caractérisées par un groupe phénotype MDR complexe, nous avons pu tester le bénéfice apporté par l'administration de leurres transcriptionnels reproduisant l'élément invMED1 sur des cellules qui coexpriment les gènes MDR1 et MVP. Ces travaux permettent d'espérer une thèrapie génique du phénotype MDR qui aboutisse au traitement des cancers résistants par la chimiothérapie conventionnelle.LYON1-BU.Sciences (692662101) / SudocSudocFranceF

Emerging Data on the Diversity of Molecular Mechanisms Involving C/D snoRNAs

Box C/D small nucleolar RNAs (C/D snoRNAs) represent an ancient family of small non-coding RNAs that are classically viewed as housekeeping guides for the 2′-O-methylation of ribosomal RNA in Archaea and Eukaryotes. However, an extensive set of studies now argues that they are involved in mechanisms that go well beyond this function. Here, we present these pieces of evidence in light of the current comprehension of the molecular mechanisms that control C/D snoRNA expression and function. From this inventory emerges that an accurate description of these activities at a molecular level is required to let the snoRNA field enter in a second age of maturity

Cloning and functional characterization of the rat α2B-adrenergic receptor gene promoter region: Evidence for binding sites for erythropoiesis-related transcription factors GATA1 and NF-E2

International audienceIn the rat, the alpha2B-adrenergic receptor (alpha2B-AR) is encoded by the rat non-glycosylated (RNG) gene and is primarily expressed in the kidney, brain and liver of adult animals. High levels of alpha2B-AR are also found during fetal life in the placenta, liver and blood, where it is borne by cells of the erythropoietic lineage. As a first step to define the mechanisms responsible for the spatio-temporal pattern of alpha2B-AR expression, a genomic fragment containing 2.8 kb of the 5'-flanking region, the ORF and approximately 20 kb of the 3'-flanking region of the RNG gene was isolated. RNase protection assays performed on RNA from placenta or kidney using a series of riboprobes permitted to locate the transcription start site 372 bases upstream from the start codon. Transient transfection of various cells, including rat proximal tubule in primary culture, with constructs containing luciferase as a reporter gene demonstrated that: (i) the 5'-flanking region exhibited a strong and sense-dependent transcriptional activity and (ii) the 332 bp fragment (-732/-401 relative to the start codon), which lacks a TATA box but contains Sp1 sites, is sufficient to drive expression. Analysis of chromatin susceptibility to DNaseI digestion identified two hypersensitive sites (HS1 and HS2) located 1.7 and 1.0 kb, respectively, upstream from ATG and containing recognition sequences for erythroid transcription factors. EMSA showed specific binding of GATA1 and NF-E2 to these elements. Taken together, the results suggest that the chromatin environment in the vicinity of these boxes plays a critical role for alpha2B-AR expression during fetal life

Major cytogenetic aberrations and typical multidrug resistance phenotype of uveal melanoma: Current views and new therapeutic prospects

International audienceUveal melanoma is the most frequent intra-ocular cancer. The recent development of new chromosome-related technologies have permitted the elucidation of both the cytogenetics and the natural history of this disease. Fifty to 60% of uveal melanomas are linked to a monosomy 3, which appears as an early and determinant event in tumor progression. Tumors with this anomaly have a very poor prognosis. Recent work suggests that this category of uveal melanoma represents a distinct pathologic entity from that associated with normal disomy 3. Chromosome 6 aberrations probably constitute a second entry point into the process of cancerogenesis, while gains in 8q seem to appear later in the natural history of uveal melanomas due to their higher frequency in larger tumors. Other anomalies will be reviewed. In spite of significant improvements in the local treatment of uveal melanoma, many patients die due to tumor metastasis. This disease is characterized by a constitutive chemoresistance whose typical multidrug resistance phenotype (MDR) is particularly complex since different combinations of several resistance proteins are simultaneously produced. Regulation of the expression of these proteins is a research priority, increasingly so as gene therapy-dependent chemosensitization strategies expand. Therefore, the development and improvement of methods to determine the chemoresistance profile become a crucial objective today in the therapeutic strategies against uveal melanoma

[Toward monosomy 3 as the main prognosis factor of uveal melanoma: current cytogenetic data].

International audienceUveal melanoma is the most frequent intraocular cancer. The recent development of new technologies such as microsatellite analysis and comparative genomic hybridization have elucidated both the cytogenetics and the natural history of this disease. Fifty to 60% of uveal melanomas are linked to monosomy 3, which appears as an early and determinant event in tumor progression. Tumors with this anomaly have a very poor prognosis. Recent work suggests that this category of uveal melanomas represents a distinct pathological entity from that associated with normal disomy 3. Chromosome 6 aberrations probably make up a second entry point into the process of carcinogenesis, while gains in 8q seem to appear later in the natural history of uveal melanoma because of their higher frequency in larger tumors. Progress in genome analysis has identified regions in chromosomes 3, 6, and 8 as those most probably involved in tumorigenesis. It is to be hoped that this will soon lead to the discovery of the genes responsible

Transcriptional regulators of the human multidrug resistance 1 gene: recent views.

International audienceThe multidrug resistance (MDR) phenotype is the major cause of failure of cancer chemotherapy. This phenotype is mainly due to the overexpression of the human MDR1 (hMDR1) gene. Several studies have shown that transcriptional regulation of this gene is unexpectedly complex and is far from being completely understood. Current work is aimed mainly at defining unclear and new control regions in the hMDR1 gene promoter as well as clarifying corresponding signaling pathways. Such studies provide new insights into the mechanisms by which xenobiotic molecules might modify the physiological hMDR1 expression as well as the possible role of oncogenes in the pathological dysregulation of the gene. Here we report recent findings on the regulation of hMDR1 which may help define specific targets aimed at modulating its transcription

Transcriptional regulation of the human MDR1 gene at the level of the inverted MED-1 promoter region.

International audienceThe typical multidrug resistance phenotype (MDR), the major cause of failure of cancer chemotherapy, is the result of the overexpression of the human MDR1 gene, the regulation of which is still incompletely understood. Using several EMSA experiments, we have identified a new regulatory sequence located from -103 to -98 bp relative to the +1 start site in the MDR1 promoter region. This sequence, which we called inverted MED-1, acts as a cis-activator for this gene. In transient transfection experiments of highly resistant human lymphoblastic CEM/VLB5 cells, its deletion from the promoter region is responsible for 60% inhibition of the MDR1 transcriptional activity. This sequence specifically binds a nuclear protein of about 150-160 kDa. We showed that its binding capacity is related to the chemoresistance level of the studied cell lines and may reflect the increased transcriptional activity of the MDR1 gene in multidrug-resistant cells

[Multidrug resistance in uveal melanoma].

International audienceIn spite of important progress in the local treatment of uveal melanoma, the most frequent primitive intraocular tumor, 15%-30% of patients still die because of tumor metastasis. This tumor is characterized by constitutive chemoresistance, thwarting any attempt to control it using the usual chemotherapy protocols. The chemoresistance of uveal melanoma is mainly due to the typical multidrug resistance phenotype (MDR), which is linked to overexpression of membrane proteins that actively extrude anticancer drugs from the cell. Typical MDR is particularly complex in this tumor since several chemoresistance-related proteins are simultaneously produced. The negative prognostic significance of the overexpression of P-glycoprotein, the main representative among the typical MDR-related proteins, was shown in uveal melanoma. The atypical MDR phenotype, which refers to other chemoresistance mechanisms such as resistance to apoptosis also contributes to the chemoresistance of uveal melanoma. Thanks to the recent progress in molecular biology, the chemosensitization strategies of gene therapy approaches, which aim at weakening the pathological activity of MDR genes in cancer cells, are currently on the rise. This approach will disrupt current therapeutic strategies and necessarily improve and standardize the methods used to characterize the chemoresistance profile of this cancer. Indeed, we will have to know the genes to be targeted for each melanoma in order to induce cell chemosensitivity

Gene Therapy of the Typical Multidrug Resistance Phenotype of Cancers: A New Hope?

International audienceThe multidrug resistance (MDR) phenotype of cancers has generated a large amount of research, owing to its constant fatal clinical outcome. Many studies have focused on the discovery of chemomodulators; however, in spite of this huge effort, the side effects that these products induce, and their additive toxicity when used in the presence of anticancer drugs, have led to the disaffection of the pharmaceutical industry and possibly slowed down research in pharmacological modulation. New tools developed using molecular biology techniques have opened the way for gene therapy and given birth to new therapeutic hopes. However, these discoveries and especially their clinical applications have slowed due to a lack of knowledge of the systems that finely regulate the MDR genes. This weakness explains why, to date, no general review has focused on the possibilities of gene therapy of MDR derived form the strategic options now available. Based on molecular foundations and recent fundamental discoveries, we seek to inform clinicians of the therapeutic hopes for chemoresistant tumors brought about by potent and specific new tools such as transcriptional decoys, interfering RNAs, etc. After describing the causes and mechanisms of MDR, we critically review these new strategies and their corresponding clinical trials