Etude des conséquences fonctionnelles de la surexpression du facteur de transcription Sp1

The Sp1 transcription factor binds the GC-rich sequences found in promoters of numerous genes. The aim of this study has been to caracterise the role of Sp1 in the regulation of apoptosis and cell cycle. We have shown that Sp1 overexpression induces a cell cycle inhibition in G1 phase and apoptosis. Cell cycle inhibition could result from the down-regulation of cyclinD2 and up-regulation of p18 and cyclinG2 as identified by expression profiling of whole genome. About mechanisms of apoptosis induction, we have shown that the apoptotic pathways activated are cell type specific and are different of those that have been already described. Unexpectedly, our results show that all these cellular perturbations require Sp1 binding to DNA but could be independent of its transcriptional activity.Le facteur de transcription Sp1 régule la transcription de nombreux gènes à partir des sites riches en GC. Mon projet a été d'étudier le rôle de Sp1 dans la régulation de l'apoptose et du cycle cellulaire, qui est encore mal défini à l'heure actuelle. Nous avons montré que la surexpression de Sp1 induit un ralentissement du cycle cellulaire en phase G1 et l'apoptose. L'étude du transcriptome a permis d'identifier les mécanismes qui pourraient être à l'origine du ralentissement du cycle cellulaire : la répression de la cycline D2 et l'induction de p18 et cycline G2. Concernant l'induction de l'apoptose, les mécanismes mis en jeu sont spécifiques du type cellulaire et sont différents de ceux décrits jusqu'à ce jour. De façon inattendue, nos résultats montrent que l'ensemble de ces perturbations cellulaires requièrent la liaison de Sp1 à l'ADN mais pourrait être indépendant de son activité transcriptionnelle

Étude des conséquences fonctionnelles de la surexposition du facteur de transcription Sp1

LYON-ENS Sciences (693872304) / SudocSudocFranceF

Overexpression of Sp1 transcription factor induces apoptosis.

International audienceTranscription factor Sp1 has recently been shown to be overexpressed in a number of human cancers and its overexpression contributes to malignant transformation. Sp1 regulates the expression of a number of genes participating in multiple aspects of tumorigenesis such as angiogenesis, cell growth and apoptosis resistance. To better understand the role of increased Sp1 levels on apoptosis regulation we have used retroviruses to overexpress this protein in haematopoietic Baf-3 cells and in 3T3 fibroblasts. We have also used inducible expression systems to control ectopic Sp1 levels in different cell types. Surprisingly, Sp1 overexpression on its own induces apoptosis in all the cellular models tested. The apoptotic pathways induced by Sp1 overexpression are cell type specific. Finally, using a truncated form of Sp1, we show that Sp1-induced apoptosis requires its DNA-binding domain. Our results highlight that Sp1 levels in untransformed cells must be tightly regulated as Sp1 overexpression leads to the induction of apoptosis. Our results also suggest that cancer cells overexpressing Sp1 can avoid Sp1-induced apoptosis

Polyaminoquinoline iron chelators for vectorization of antiproliferative agents: design, synthesis, and validation.

International audienceIron chelation in tumoral cells has been reported as potentially useful during antitumoral treatment. Our aim was to develop new polyaminoquinoline iron chelators targeting tumoral cells. For this purpose, we designed, synthesized, and evaluated the biological activity of a new generation of iron chelators, which we named Quilamines, based on an 8-hydroxyquinoline (8-HQ) scaffold linked to linear polyamine vectors. These were designed to target tumor cells expressing an overactive polyamine transport system (PTS). A set of Quilamines bearing variable polyamine chains was designed and assessed for their ability to interact with iron. Quilamines were also screened for their cytostatic/cytotoxic effects and their selective uptake by the PTS in the CHO cell line. Our results show that both the 8-HQ moiety and the polyamine part participate in the iron coordination. HQ1-44, the most promising Quilamine identified, presents a homospermidine moiety and was shown to be highly taken up by the PTS and to display an efficient antiproliferative activity that occurred in the micromolar range. In addition, cytotoxicity was only observed at concentrations higher than 100 μM. We also demonstrated the high complexation capacity of HQ1-44 with iron while much weaker complexes were formed with other cations, indicative of a high selectivity. We applied the density functional theory to study the binding energy and the electronic structure of prototypical iron(III)-Quilamine complexes. On the basis of these calculations, Quilamine HQ1-44 is a strong tridentate ligand for iron(III) especially in the form of a 1:2 complex

Click-electrochemistry for the rapid labeling of virus, bacteria and cell surfaces

The remodeling of microorganism surfaces with biomolecules is a powerful tool to study the role of membrane receptors in chemical biology and to develop drug delivery systems in gene therapy using viral vectors and cell-based therapies. Methods for direct covalent ligation of these surfaces remain poorly reported, and mostly based on metabolic engineering for bacteria and cells functionalization. In the latter case, a tagged precursor must first be enzymatically metabolized and delivered to the outer cell membrane to become available for chemo-selective labeling. While effective, a faster method avoiding the bio-incorporation step would be highly complementary. This would also need to be compatible with organisms showing poor levels of precursor assimilation or lacking the metabolic function. Here, we used N-methylluminol (NML), a fully tyrosine-selective protein anchoring group after one-electron oxidation, to label the surface of viruses, living bacteria and cells. The functionalization was performed electrochemically and in situ by applying a 750 mV vs Ag/AgCl electric potential to aqueous buffered solutions of tagged NML containing the viruses, bacteria or cells. The electro-coupling was performed with NML anchors bearing a bioorthogonal azide, biotin, or carbohydrate (mannose and N-acetyl galactosamine) handles. The broad applicability of the click-electrochemistry method was explored on recombinant adeno-associated viruses (rAAV2), E. coli (Gram-) and S. epidermis (Gram+) bacterial strains, and HEK293 and HeLa eukaryotic cell lines. Surface electro-conjugation was achieved in minutes to yield functionalized rAAV2 that conserved both structural integrity and infectivity properties, and living bacteria and cell lines that were still alive and able to divide. As NML activation immediately stops if there is no current, the method offers reproducible temporal control on the degree of surface functionalization. Thus, click-electrochemistry should significantly expand the scope of bioconjugation methods

Click-electrochemistry for the rapid labeling of virus, bacteria and cell surfaces

Abstract Methods for direct covalent ligation of microorganism surfaces remain poorly reported, and mostly based on metabolic engineering for bacteria and cells functionalization. While effective, a faster method avoiding the bio-incorporation step would be highly complementary. Here, we used N-methylluminol (NML), a fully tyrosine-selective protein anchoring group after one-electron oxidation, to label the surface of viruses, living bacteria and cells. The functionalization was performed electrochemically and in situ by applying an electric potential to aqueous buffered solutions of tagged NML containing the viruses, bacteria or cells. The broad applicability of the click-electrochemistry method was explored on recombinant adeno-associated viruses (rAAV2), Escherichia coli (Gram-) and Staphyloccocus epidermidis (Gram + ) bacterial strains, and HEK293 and HeLa eukaryotic cell lines. Surface electro-conjugation was achieved in minutes to yield functionalized rAAV2 that conserved both structural integrity and infectivity properties, and living bacteria and cell lines that were still alive and able to divide

Overexpression of the Transcription Factor Sp1 Activates the OAS-RNAse L-RIG-I Pathway

<div><p>Deregulated expression of oncogenes or transcription factors such as specificity protein 1 (Sp1) is observed in many human cancers and plays a role in tumor maintenance. Paradoxically in untransformed cells, Sp1 overexpression induces late apoptosis but the early intrinsic response is poorly characterized. In the present work, we studied increased Sp1 level consequences in untransformed cells and showed that it turns on an early innate immune transcriptome. Sp1 overexpression does not activate known cellular stress pathways such as DNA damage response or endoplasmic reticulum stress, but induces the activation of the OAS-RNase L pathway and the generation of small self-RNAs, leading to the upregulation of genes of the antiviral RIG-I pathway at the transcriptional and translational levels. Finally, Sp1-induced intrinsic innate immune response leads to the production of the chemokine CXCL4 and to the recruitment of inflammatory cells <i>in vitro</i> and <i>in vivo</i>. Altogether our results showed that increased Sp1 level in untransformed cells constitutes a novel danger signal sensed by the OAS-RNase L axis leading to the activation of the RIG-I pathway. These results suggested that the OAS-RNase L-RIG-I pathway may be activated in sterile condition in absence of pathogen.</p></div

Sp1 activates the OAS-RNAse L pathway and the production of small self-RNAs.

<p>(A) BaF3-Sp1 inducible cell line was grown with (white bars) or without (black bars) dox for the indicated times, and relative mRNA levels of OAS2 and RNase L genes were quantified by RT-qPCR. (B, D, E) 3T3 (B) and MEFs (D, E) WT or deficient for the RNase L (RNase L KO) or MAVS (MAVS KO) were transduced with Sp1, Sp1 Zn^2,3 (Zn) or empty vector (EV). Transduced cells (CD2 positive) were purified 72 h post-transduction by magnetic selection and relative mRNA levels of indicated genes were quantified by RT-qPCR. (C) Small RNAs from BaF3-Sp1 cells following induction Sp1 or not were extracted at the indicated times, and transfected into 293HEK cells (5μg per condition). Luciferase reporter assay was performed to analyze the ISRE promoter activity. Data are mean values ± standard deviation (SD) from one experiment representative of two or three independent experiments. Statistical analysis were performed using 2-tailed <i>t</i> tests. Levels of significance are expressed as follows: *<i>P</i> <0.05; **<i>P</i> <0.01.</p

Functional analysis of Sp1 signature.

<p>The gene list enrichment analysis from the Gene Ontology of the SP1 specific signature was performed with g:Profiler. The moderate hierarchical filtering used here allows a compact representation of gene list enrichment results. Significantly enriched GO terms containing less than 5 genes were excluded.</p><p>Functional analysis of Sp1 signature.</p