Transcriptional networks of lung airway epithelial ciliogenesis

Motile cilia of the mammalian airway play an essential role in innate defense. The coordinated transcriptional regulation of cilial axoneme genes remains to be elucidated. Transcription factor FOXJ1 has been shown to be important in ciliogenesis; however, direct transactivation of cilia genes by FOXJ1 has not been reported. Using a combined bioinformatics and experimental approach, here, we show a transcriptional network for cilia gene expression. FOXJ1 can directly transactivate endogenous cilia genes such as ENKURIN, EFHC2, IFT57, RIBC2 and ROPN1L in human bronchial epithelial cells (HBEC). FOXJ1 transactivation is localized to the proximal end of the 5 flanking region of ENKURIN and EFHC2 promoters. However, FOXJ1 failed to transactivate HSPA1A and MNS1, two other cilia genes, indicating there are other transcription factor(s)involved in the ciliogenesis. Motif discovery analysis indicates ETS and RFX binding sites located in promoters of several cilia genes (IFT57, HSPA1A, MNS1, RIBC2 and ROPN1L). QRT-PCR indicates 5 of 27 human ETS transcription factors members (ETV1, ETV5, SPDEF, SPIC and ESE1) and 4 of 7 RFX transcription factors (RFX1, RFX2, RFX5 and RFXANK) are regulated coincidently with the differentiation of human bronchial epithelial cells and the appearance of cilia, suggesting these transcription factors are involved in gene regulations during lung airway epithelial ciliogenesis. ETS family members ESE1 and ETV1 both can transactivate endogenous EFHC2 and ENKURIN while SPIC represses EFHC2. An RFX family member RFX1 transactivates endogenous ENKURIN, HSPA1A, ROPN1L, and RFX5 transactivates EFHC2, HSPA1A and RIBC2. Furthermore, another transcription factor, GATA2, transactivates endogenous ENKURIN and EFHC2, and its transactivation also locates in the proximal end of the 5\u27 flanking region of ENKURIN and EFHC2 promoters. Our work defines a transcriptional network that regulates expression of cilia genes during airway epithelial differentiation and highlights the importance of multiple transcription factors in ciliogenesis

Molecular analysis of the domain with no name (DWNN)/RBBP6 in human cancers

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. It is a multi-domain protein containing a Zinc finger, a RING finger, an Rb binding domain, a p53 binding domain and a novel N-terminal protein domain, the so called, Domain With No Name or DWNN. The RBBP6 gene encodes three isoforms of this particular protein. A common feature of all three isoforms of RBBP6 is the presence of the N-terminal DWNN domain. RBBP6 isoform 3 is comprised of the DWNN domain only. The DWNN itself has a ubiquitin-like fold, sharing 22% similarity with ubiquitin. It is likely that DWNN regulates intracellular levels of the two tumour suppressors, Rb and p53 through the ubiquitin-proteasome pathway and as such, DWNN may therefore play a role in the deregulation of cell cycle control in cancer cells. A mouse homologue, P2P-R of the gene has been implicated in mitotic apoptosis. The expression of DWNN, RBBP6 and their roles in the cell cycle, apoptosis and human cancer were investigated. RT-PCR and real-time PCR were used to determine the gene expression of DWNN and RBBP6 variants in human cancer cells. An anti-human DWNN antibody was characterized using both Western Blotting analysis and MALDI-TOF mass spectroscopy to determine whether the antibody specifically recognizes DWNN and RBBP6 isoforms, or if it recognizes other proteins. Western blotting was also used to determine the nature of the DWNN in human cell lines. A DWNN probe and the characterized anti-human antibody were used to localize DWNN and RBBP6 gene products at the mRNA and protein levels using ISH/FISH and Immunohistochemistry respectively. Cell labelling was also performed using this antibody to localize RBBP6 products in human cell lines. RNA interference and over-expression of DWNN and RBBP6 gene products was carried out to further investigate the role of RBBP6 products in the cell cycle, apoptosis and carcinogenesis. Cloned RT-PCR products of RBBP6 binding domains, the RING finger domain, pRb-binding and p53-binding domains in human cancers cell lines (Hek 293T, MCF7, HeLa and HepG2 cells) showed no mutations, but MCF-7 cells showed the lowest expression of the RBBP6. Real-time PCR and Western blotting analysis confirmed that MCF-7 cells express very little DWNN (RBBP6 isoform 3) and RBBP6 gene products when compared to Hek 293T, HeLa and HepG2 cells. It was also shown that the anti-human DWNN antibody recognizes the DWNN domain (RBBP6 isoform 3) and the larger RBBP6 isoforms. Using 2D gel electrophoresis and MALDI-TOF spectrometry, it was also found that DWNN is associated with other proteins namely, Recoverin and a hypothetical protein XP_002342450. This result suggested that DWNN may be a ubiquitin-like protein, which may be specific to these proteins in human cells. FISH and IHC demonstrated that the DWNN domain and its relatives are down-regulated in human cancers at both mRNA and protein levels, respectively. In contrast, however, cell staining showed that the expression of the DWNN gene products was high during the G2/Mitosis transition. Knocking-down the DWNN domain or over-expressing it did not sensitise the Hek 293T cells to Camptothecin (CPT)-induced apoptosis but rather slowed down cell growth. These results strongly suggest that the DWNN gene is likely to be involved in cell cycle control. Up-regulation in mitotic cells and down-regulation in cancers also implies that RBBP6 gene products may additionally be involved in cell cycle arrest. Moreover, down-regulation in human cancers particularly indicates that the loss of its function which correlates with loss of cell cycle control in this disease may be involved in the pathogenesis of cancer. This was confirmed by up-regulation of the DWNN in arsenic trioxide induced cell cycle arrested cells specifically at G2/M phase where a p53-dependent cell cycle arrest ensued. It is thus proposed that the DWNN may be implicated both as a p53 stabilizer and additionally as a G2/M progression regulator

Localizing potentially active post-transcriptional regulations in the Ewing's sarcoma gene regulatory network.

BACKGROUND: A wide range of techniques is now available for analyzing regulatory networks. Nonetheless, most of these techniques fail to interpret large-scale transcriptional data at the post-translational level. RESULTS: We address the question of using large-scale transcriptomic observation of a system perturbation to analyze a regulatory network which contained several types of interactions - transcriptional and post-translational. Our method consisted of post-processing the outputs of an open-source tool named BioQuali - an automatic constraint-based analysis mimicking biologist's local reasoning on a large scale. The post-processing relied on differences in the behavior of the transcriptional and post-translational levels in the network. As a case study, we analyzed a network representation of the genes and proteins controlled by an oncogene in the context of Ewing's sarcoma. The analysis allowed us to pinpoint active interactions specific to this cancer. We also identified the parts of the network which were incomplete and should be submitted for further investigation. CONCLUSIONS: The proposed approach is effective for the qualitative analysis of cancer networks. It allows the integrative use of experimental data of various types in order to identify the specific information that should be considered a priority in the initial - and possibly very large - experimental dataset. Iteratively, new dataset can be introduced into the analysis to improve the network representation and make it more specific

Optical-guided surgery of the feline fibrosarcoma & Development and characterization of a bi-functional vector for cancer targeting

Actuellement, la chirurgie représente la première indication pour la thérapie du cancer. Néanmoins, la résection complète du tissu tumoral, la détection des micrométastases et la préservation des tissus sains pendant l'intervention représentent un enjeu majeur et influencent fortement le pronostic du patient. Les récents développements technologiques en imagerie pour la chirurgie guidée des cancers ont conduit à des résultats précliniques prometteurs et les premiers essais cliniques utilisant des traceurs non-spécifiques confirment déjà le potentiel de ces systèmes pour l'amélioration de la chirurgie. De plus, le diagnostic précoce des tumeurs, ainsi que le développement de thérapies ciblées sont également des axes majeurs de recherche en cancérologie. Dans ce contexte notre équipe a précédemment développé un vecteur synthétique ciblant un récepteur cellulaire l'intégrine aVb3. Ce vecteur est constitué d'un châssis décapeptidique cyclique RAFT (Regioselectively Addressable Functionalized Template) et présentant deux domaines indépendants permettant de séparer les deux fonctions du vecteur. Sur un domaine, la fonction de ciblage est assurée par la présentation multivalente de ligands -RGD- spécifiques du récepteur. L'autre domaine du vecteur porte les molécules d'intérêt à vectoriser, agents thérapeutiques ou de détection pour l'imagerie médicale. Dans la première partie de ces travaux, nous avons évalué la combinaison de ce vecteur couplé à un fluorophore avec une sonde portative pour imager et guider le chirurgien pendant la chirurgie des fibrosarcomes spontanés chez le chat. Cette étude représente une preuve de concept pour la translation clinique chez l'homme. Les résultats ont montré que l'injection du traceur ne provoquait pas d'effets toxiques chez le chat et permettait un marquage spécifique de la tumeur avec un bon ratio tumeur/tissu sain, qui devrait améliorer la qualité de la résection tumorale en aidant le chirurgien à mieux délimiter les marges du tissu tumoral. Dans la seconde partie de ces travaux nous avons développé un nouveau vecteur bi-fonctionnel dérivé du RAFT-RGD. Au composé d'origine a été ajoutée une séquence peptidique clivable par la matrixmetalloprotease-9, une enzyme surexprimée dans la tumorigénèse. Cette molécule à fluorescence activable a montré une amélioration du ciblage tumoral in vitro et in vivo comparée au RAFT-RGD suggérant un effet additionnel lié au double ciblage. Ces résultats préliminaires encouragent la poursuite de sa caractérisation pour son potentiel de pro-drug mais également pour l'étude des interactions entre l'intégrine et l'environment tumoraux.Cancer surgery is still the gold standard therapy in most cancers. Nevertheless, total tumor resection and metastasis detection while preserving healthy tissues represent a crucial point for further prognosis. Development of imaging technologies for intra-operative guided surgery provided promising results and efficient application in preclinical studies and first clinical trials using non-specific tracers already confirmed the improved out-come in surgery. Moreover early and precise diagnosis and targeted therapies are major domains of cancer research. In this context our team previously developed a synthetic vector based on a cyclic decapeptide scaffold RAFT (Regioselectively Addressable Functionalized Template) which allows the independent functionalizing of two domains: a targeting domain with multivalent RGD-ligand targeting the cell receptor integrin aVb3, and a vehicle domain grafted with a pro-drug or an imaging agent. One part of this work consisted in the evaluation of the combination of this molecule carrying a fluorophore with a portable fluorescent imaging device for image-guided surgery of natural occurring feline fibrosarcomas. This study represents a proof of concept for further translation into human clinics. No toxic effects in cats after administration of the tracer could be reported. Furthermore the tumors were specifically labeled showing a good tumor-to-healthy tissue ratio. This should improve tumor resection by helping the surgeon to delineate tumor margins. In parallel we developed a bi-functinal derivative of the RAFT-RGD. Therefore we engrafted a peptide sequence flanked by two fluorophores, which is activatable by matrixmetalloprotease-9, an enzyme overexpressed in tumors. This molecule showed an improved tumor labeling in vitro and in vivo compared to the conventional RAFT-RGD, suggesting an additional effect of the double targeting. These preliminary results encourage further caracterisation for its potential as pro-drug vehicle, as well as for studying interactions between the integrin and the tumor environment.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Biocompatible tumour implant systems: towards an integrated biophotonic system

There is a need to perform comprehensive cell biology studies transferable across culture platforms using innovative cellular models. The higher purpose is to bridge the gap between in vitro cell culture and in vivo models. In this thesis a significant advance is presented in the embedding of an innovative optical biophotonic capability for the dynamic interrogation and single cell tracking of human osteosarcoma cells encapsulated in the hollow fiber (HF) platform. Two approaches have been implemented: quantum dot (QD) nanoparticles providing proliferative and cell cycle readouts and an in-fiber light illumination providing global features of particle and cell density. An in vitro HF encapsulation model was developed and characterised against standard two-dimensional tissue culture (TC) using the human osteosarcoma U-2 OS cell line expressing a cell cycle fluorescent reporter (cyclin Bl-GFP). Analysis of the packing and orientation of cells in the HF revealed that they grow like an anchorage dependent adherent layer. Overall cells in the fiber displayed a slower cell cycle traverse and a differential sensitivity to clinically relevant doses of the anticancer mitosis-inhibiting agent Taxol compared to cells under normal TC conditions. Comprehensive gene profiling, with bioinformatics and ontology network analysis, showed that the HF cells presented high steroid related but low differentiation gene expression. Specific biomarkers were indentified, and it is suggested that the HF model displays features that are closer to an in vivo tumour. A flow cytometry cell-tracking approach using QD labelling was validated and applied to the HF model for the first time. This represents an "embedded" biophotonic system where the QD sensors are integrated directly into the seeded cell population and then redistributed through the daughter cells, thus reflecting patterns of lineage expansion. This provides sub-population parameterized information on cell-cell heterogeneity and cell division. A biophotonic HF prototype comprising the integration of direct coupled-light excitation in the HF was conceived, this revealed the potential and limitations to detect die presence of cells inside the HF lumen by analysing light attenuation changes. Finally a "systems cytometry" acquisition concept has been proposed, comprising the use of embedded engineered nanoparticles as single cell "nano-memory" biophotonic intracellular probes