Fabrice Magnino
@illumina.com
Illumina
RESEARCH INTERESTS
Oncology and Virology
Scopus Publications
Scopus Publications
Christophe Côme, Fabrice Magnino, Frédéric Bibeau, Pascal De Santa Barbara, Karl Friedrich Becker, Charles Theillet, and Pierre Savagner
American Association for Cancer Research (AACR)
Purpose: Carcinoma progression is linked to a partially dedifferentiated epithelial cell phenotype. As previously suggested, this regulation could involve transcription factors, Snail and Slug, known to promote epithelial-mesenchymal transitions during development. Here, we investigate the role of Snail and Slug in human breast cancer progression. Experimental Design: We analyzed Snail, Slug, and E-cadherin RNA expression levels and protein localization in large numbers of transformed cell lines and breast carcinomas, examined the correlation with tumor histologic features, and described, at the cellular level, Snail and Slug localization in carcinomas using combined in situ hybridization and immunolocalization. Results: In contrast with transformed cell lines, Slug was found to colocalize with E-cadherin at the cellular level in normal mammary epithelial cells and all tested carcinomas. Snail also colocalized at the cellular level with E-cadherin in tumors expressing high levels of Snail RNA. In addition, Snail was significantly expressed in tumor stroma, varying with tumors. Slug and Snail genes were significantly overexpressed in tumors associated with lymph node metastasis. Finally, the presence of semidifferentiated tubules within ductal carcinomas was linked to Slug expression levels similar to or above normal breast samples. Conclusions: These results suggest that Snail or Slug expression in carcinoma cells does not generally preclude significant E-cadherin expression. They emphasize a link between Snail, Slug, and lymph node metastasis in a large sampling of mammary carcinomas, and suggest a role for Slug in the maintenance of semidifferentiated structures. Snail and Slug proteins seem to support distinct tumor invasion modes and could provide new therapeutic targets.
Juliette Martin, Fabrice Magnino, Karin Schmidt, Anne–Christine Piguet, Ju–Seog Lee, David Semela, Marie V. St–Pierre, Andrew Ziemiecki, Doris Cassio, Charles Brenner,et al.
Elsevier BV
BACKGROUND & AIMS
Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2.
METHODS
Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays.
RESULTS
Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 +/- 0.13 g vs 0.85 +/- 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (-0.42 +/- 0.58 log2 vs -0.11 +/- 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival.
CONCLUSION
Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.
Pierre Savagner, Donna F. Kusewitt, Ethan A. Carver, Fabrice Magnino, Chagsun Choi, Thomas Gridley, and Laurie G. Hudson
Wiley
During re‐epithelialization of cutaneous wounds, keratinocytes recapitulate several aspects of the embryonic process of epithelial‐mesenchymal transition (EMT), including migratory activity and reduced intercellular adhesion. The transcription factor Slug modulates EMT in the embryo and controls desmosome number in adult epithelial cells, therefore, we investigated Slug expression and function during cutaneous wound re‐epithelialization. Slug expression was elevated in keratinocytes bordering cutaneous wounds in mice in vivo, in keratinocytes migrating from mouse skin explants ex vivo, and in human keratinocytes at wound margins in vitro. Expression of the related transcription factor Snail was not significantly modulated in keratinocytes during re‐epithelialization in vitro. Epithelial cell outgrowth from skin explants of Slug knockout mice was severely compromised, indicating a critical role for Slug in epithelial keratinocyte migration. Overexpression of Slug in cultured human keratinocytes caused increased cell spreading and desmosomal disruption, both of which were most pronounced at wound margins. Furthermore, in vitro wound healing was markedly accelerated in keratinocytes that ectopically expressed Slug. Taken together, these findings suggest that Slug plays an important role during wound re‐epithelialization in adult skin and indicate that Slug controls some aspects of epithleial cell behavior in adult tissues as well as during embryonic development. © 2004 Wiley‐Liss, Inc.
JA Vendrell, F Magnino, E Danis, MJ Duchesne, S Pinloche, M Pons, D Birnbaum, C Nguyen, C Theillet, and PA Cohen
Bioscientifica
We explored, by cDNA mini-arrays, gene expression measurements of MVLN, a human breast carcinoma cell line derived from MCF-7, after 4 days of exposure to 17beta-estradiol (E(2)) treatment, in order to extend our understanding of the mechanism of the pharmacological action of estrogens. We focused on 22 genes involved in estrogen metabolism, cell proliferation regulation and cell transformation. The specificity of the E(2) response was reinforced by comparison with 4-hydroxytamoxifen (OH-Tam), ICI 182,780 and E(2)+OH-Tam expression profiles. Real-time quantitative PCR (RTQ-PCR) confirmed the variation of expression of known (TFF1, AREG, IRS1, IGFBP4, PCNA, ERBB2, CTSD, MYC) as well as novel (DLEU2, CCNA2, UGT1A1, ABCC3, ABCC5, TACC1, EFNA1, NOV, CSTA, MMP15, ZNF217) genes. The temporal response of these gene expression regulations was then investigated after 6 and 18 h of E(2) treatment and this allowed the identification of different time-course patterns. Cycloheximide treatment studies indicated first that estrogen affected the transcript levels of ABCC3 and ABCC5 through dissimilar pathways, and secondly that protein synthesis was needed for modulation of the expression of the CCNA2 and TACC1 genes by estrogens. Western blot analysis performed on TFF1, IRS1, IGFBP4, amphiregulin, PCNA, cyclin A2, TACC1 and ABCC5 proteins confirmed the mini-array and RTQ-PCR data, even for genes harboring low variations of mRNA expression. Our findings should enhance the understanding of changes induced by E(2) on the transcriptional program of human E(2)-responsive cells and permit the identification of new potential diagnostic/prognostic tools for the monitoring of estrogen-related disease conditions such as breast cancer.
Fabrice Magnino, Karin Schmidt, Laurence Mery, and Jean-François Dufour
Wiley
In response to stimulation at the plasma membrane, hepatocellular Ca(2+) signals are fast and precise and lead to rapid local changes in cytoplasmic free Ca(2+) concentration. These changes result from the opening of the inositol 1,4,5-trisphosphate receptor (InsP(3)R), which is a four-subunit intracellular InsP(3)-gated channel that releases Ca(2+) from the stores. To investigate the molecular mechanism underlying interactions between the InsP(3)R subunits, we cloned the predominant hepatocellular isoform, InsP(3)R isoform 2 (InsP(3)R2), and screened for interactions using the yeast two-hybrid assay. We found that the C-terminal domain of rat InsP(3)R2 interacts with itself, and that the cytoplasmic part preceding the first transmembrane domain, a region near a Ca(2+)-binding site, also interacts with itself. These interactions were confirmed by pull-down experiments. The C-terminal domain of InsP(3)R2 is also able to interact with the C-termini of rat InsP(3)R1 and InsP(3)R3. These results advance our understanding of the molecular mechanisms that underlie the oligomerization and interactions of the InsP(3)R subunits during the opening/closing of the Ca(2+) channel.
Laurence Mery, Fabrice Magnino, Karin Schmidt, Karl-Heinz Krause, and Jean-François Dufour
Wiley
The molecular basis of capacitative (or store‐operated) Ca2+ entry is still subject to debate. The transient receptor potential proteins have been hypothesized to be structural components of store‐operated Ca2+ channels and recent evidence suggests that Trp3 and its closely related homolog Trp6 are gated by the N‐terminal region of the inositol 1,4,5‐triphosphate receptors (InsP3R). In this study, we report the existence of two isoforms of the human Trp4 protein, referred to as α‐hTrp4 and β‐hTrp4. The shorter variant β‐hTrp4 is generated through alternative splicing and lacks the C‐terminal amino acids G785–S868. Using a yeast two‐hybrid assay and glutathione‐S‐transferase‐pulldown experiments, we found that the C‐terminus of α‐hTrp4, but not of β‐hTrp4, associates in vitro with the C‐terminal domain of the InsP3 receptors type 1, 2 and 3. Thus, we describe a novel interaction between Trp proteins and InsP3R and we provide evidence suggesting that the formation of hTrp4–InsP3R complexes may be regulated by alternative splicing.
Fabrice Magnino, Marie St-Pierre, Michael Lüthi, Mauricette Hilly, Jean-Pierre Mauger, and Jean-François Dufour
Elsevier BV
Ca(2+) signals regulate many cellular functions, including proliferation. They are governed by the inositol 1,4,5-trisphosphate receptor (IP(3)R), the only intracellular hepatic Ca(2+) channel and by the endoplasmic reticulum Ca(2+) pumps, SERCA. To characterise their role in regeneration, expression of their isoforms was studied after 2/3 hepatectomy by real-time quantitative PCR, Western blot and binding studies. We found an early increase in the expression of the IP(3)R isoform 1 which contrasted with the decrease of the expression of the IP(3)R isoforms 2 and 3 and of SERCA3. This results in a transient switch between IP(3)R isoforms 1 and 2, IP(3)R isoform 1 becoming predominant before the first round of mitosis. Binding studies detected a 30% diminution of the IP(3)R population at 24 h. In conclusion, the Ca(2+) signalling machinery is regulated, after hepatectomy, by changes in expression of the IP(3)R and SERCA isoforms to adapt Ca(2+) signals to the regenerative state.
Jean-François Dufour, Michael Lüthi, Marc Forestier, and Fabrice Magnino
Wiley
Ca2+ signals mediate the hepatic effects of numerous hormones and growth factors. Hepatic Ca2+ signals are elicited by the inositol trisphosphate receptor, an intracellular Ca2+ channel. Three isoforms of this receptor have been identified; they are expressed and regulated differently. We investigated the effect of liver fibrosis and cirrhosis on the hepatic expression of the inositol trisphosphate receptor isoforms. Two different rat models were used: bile duct ligation (fibrosis) and chronic exposure to CCl4/phenobarbital (cirrhosis). Messenger RNA levels were determined by ribonuclease protection assay (RPA), competitive polymerase chain reaction (PCR) followed by Southern blotting, and real‐time quantitative PCR. Protein expression was assessed by Western blotting; tissue distribution was assessed by immunohistology. In control animals, isoform 2 was the predominant isoform, isoform 1 represented less than one third, and isoform 3 less than 1%. After bile duct ligation, expression of types 1 and 3 increased 1.9‐ and 5.7‐fold, and expression of type 2 decreased 2.5‐fold at the protein level. After exposure to CCl4/phenobarbital, expression of types 1, 2, and 3 were 2.4‐, 0.9‐, and 4.2‐fold their expression in control animals. Type 2 was localized to the apical domain of hepatocytes, consistent with a role for Ca2+ signals in canalicular function. Type 3 was detectable in intrahepatic bile duct epithelial cells and not in hepatocytes, suggesting that Ca2+ signals may be regulated differently in these cells. Signaling through inositol trisphosphate receptor participates in the pathogenesis of cirrhosis, because this process affects the expression of its isoforms.
Catherine Chapelin, Bénédicte Duriez, Fabrice Magnino, Michel Goossens, Estelle Escudier, and Serge Amselem
Wiley
Dynein heavy chains (DHCs) are the main components of multisubunit motor ATPase complexes called dyneins. Axonemal dyneins provide the driving force for ciliary and flagellar motility. Recent molecular studies demonstrated that multiple DHC isoforms are produced by separate genes. We describe the isolation of five human axonemal DHC genes. Analysis of the human genomic clones revealed the existence of intronic sequences that were used to demonstrate that human axonemal DHC genes are located on different chromosomes. The cloned human DHC sequences were integrated into an evolutionary approach based on phylogenetic analysis. Tissue expression studies showed that these human axonemal DHCs are expressed in testis and/or trachea, two tissues with axonemal structures that can be altered in primary ciliary dyskinesia, making DHC genes strong candidates in the genesis of these human diseases.