Cellular and molecular characterization of IDH1-mutated diffuse low grade gliomas reveals tumor heterogeneity and absence of EGFR/PDGFRα activation.

Diffuse low grade gliomas (DLGG, grade II gliomas) are slowly-growing brain tumors that often progress into high grade gliomas. Most tumors have a missense mutation for IDH1 combined with 1p19q codeletion in oligodendrogliomas or ATRX/TP53 mutations in astrocytomas. The phenotype of tumoral cells, their environment and the pathways activated in these tumors are still ill-defined and are mainly based on genomics and transcriptomics analysis. Here we used freshly-resected tumors to accurately characterize the tumoral cell population and their environment. In oligodendrogliomas, cells express the transcription factors MYT1, Nkx2.2, Olig1, Olig2, Sox8, four receptors (EGFR, PDGFRα, LIFR, PTPRZ1) but not the co-receptor NG2 known to be expressed by oligodendrocyte progenitor cells. A variable fraction of cells also express the more mature oligodendrocytic markers NOGO-A and MAG. DLGG cells are also stained for the young-neuron marker doublecortin (Dcx) which is also observed in oligodendrocytic cells in nontumoral human brain. In astrocytomas, MYT1, PDGFRα, PTPRZ1 were less expressed whereas Sox9 was prominent over Sox8. The phenotype of DLGG cells is overall maintained in culture. Phospho-array screening showed the absence of EGFR and PDGFRα phosphorylation in DLGG but revealed the strong activation of p44/42 MAPK/ERK which was present in a fraction of tumoral cells but also in nontumoral cells. These results provide evidence for the existence of close relationships between the cellular phenotype and the mutations found in DLGG. The slow proliferation of these tumors may be associated with the absence of activation of PDGFRα/EGFR receptors.

Transformation Foci in IDH1-mutated Gliomas Show STAT3 Phosphorylation and Downregulate the Metabolic Enzyme ETNPPL, a Negative Regulator of Glioma Growth.

IDH1-mutated gliomas are slow-growing brain tumours which progress into high-grade gliomas. The early molecular events causing this progression are ill-defined. Previous studies revealed that 20% of these tumours already have transformation foci. These foci offer opportunities to better understand malignant progression. We used immunohistochemistry and high throughput RNA profiling to characterize foci cells. These have higher pSTAT3 staining revealing activation of JAK/STAT signaling. They downregulate RNAs involved in Wnt signaling (DAAM2, SFRP2), EGFR signaling (MLC1), cytoskeleton and cell-cell communication (EZR, GJA1). In addition, foci cells show reduced levels of RNA coding for Ethanolamine-Phosphate Phospho-Lyase (ETNPPL/AGXT2L1), a lipid metabolism enzyme. ETNPPL is involved in the catabolism of phosphoethanolamine implicated in membrane synthesis. We detected ETNPPL protein in glioma cells as well as in astrocytes in the human brain. Its nuclear localization suggests additional roles for this enzyme. ETNPPL expression is inversely correlated to glioma grade and we found no ETNPPL protein in glioblastomas. Overexpression of ETNPPL reduces the growth of glioma stem cells indicating that this enzyme opposes gliomagenesis. Collectively, these results suggest that a combined alteration in membrane lipid metabolism and STAT3 pathway promotes IDH1-mutated glioma malignant progression.

Nuclear localization of the testis determining gene product SRY.

We have studied the expression of the human SRY protein (termed p27SRY) in two different cell lines by using specific antibodies. Confocal microscopy enabled us to localize p27SRY precisely in the nucleus in a discrete punctuate pattern. Furthermore, through microinjection experiments, we have demonstrated that the localization of the p27SRY protein into the nucleus was an event involving the NH2-terminal part of the high mobility group (HMG) domain. With the help of several synthetic peptides and various p27SRY mutants, we have characterized a bipartite basic motif in this part of the protein corresponding to a nuclear localization signal. This nuclear localization signal appears to be highly conserved in SRY box- and HMB box-containing proteins, suggesting common properties of nuclear targeting within the HMG box protein family.

[Molecular pharmacogenetics in hospital laboratories in France: current data and future prospects]. / La pharmacogénétique moléculaire hospitalière en France: données actuelles et perspectives.

Molecular pharmacogenetic units have recently been established in several hospital laboratories in France. The clinical impact of these units is still limited and numerous problems of organizational, ethical, legal, technical, social and economical nature remain to be resolved. However, an increasing number of these units, a rise in their activities and an enlargement of their scope of application are foreseeable in the future. Ultimately, these units would significantly contribute to limit the public health problem caused by interindividual variabilities in drug effects. In view of these prospects, it seems essential that such hospital activity should be quickly recognised by the authorities and the various health sectors in France. It is also essential that the problems that arise from such pharmacogenetic activities should be considered by the authorities and would profit from the organization of a national network and from financial guarantees.

MANOTA: a promising bifunctional chelating agent for copper-64 immunoPET.

Improved bifunctional chelating agents (BFC) are required for copper-64 radiolabelling of monoclonal antibodies (mAbs) under mild conditions to yield stable, target-specific imaging agents. Four different bifunctional chelating agents (BFC) were evaluated for Fab (Fragment antigen binding) conjugation and radiolabelling with copper-64. Two DOTA- (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and two NOTA- (1,4,7-triazacyclononane-1,4,7-triacetic acid) derivatives bearing a p-benzyl-isothiocyanate group were conjugated to Fab-trastuzumab - which targets the HER2/neu receptor - and the average number of chelators attached ranged from 2.4 to 4.3 macrocycles per Fab. Labelling of the immunoconjugate with copper-64 was achieved in high radiochemical yields after 45 min at 37 °C, and the radiochemical purity of each 64Cu-BFC-Fab-trastuzumab reached 97% after purification. The affinity of each 64Cu-BFC-Fab-trastuzumab ranged between 10 and 50 nM as evaluated by in vitro saturation assays using the HCC1954 breast cancer cell line. PET-MR imaging and biodistribution studies were performed in mice bearing breast cancer BT-474 xenografts. BT-474 tumours were clearly visualized on PET images at 4 and 24 hours post-injection. The tumour uptake of 64Cu-BFC-Fab-trastuzumab reached 8.9 to 12.8% ID g-1 24 hours post-injection and significant differences in non-specific liver uptake were observed depending on the BFC conjugated, the lowest being observed with MANOTA. These results show that MANOTA is a valuable tool for copper-64 radiolabelling.

Serum level of 7 alpha-hydroxycholesterol in hypercholesterolemic patients treated with cholestyramine.

Serum levels of 7 alpha-hydroxycholesterol (7 alpha (OH)C) were found to increase considerably in hypercholesterolemic patients treated with cholestyramine. After 3-5 days, the normal level, which is in the range of 0.04 mg/l, rose to 1 mg/l. When the treatment was stopped it decreased rapidly with a half-life analogous to that of LDLs. This phenomenon was also observed in non-hypercholesterolemic humans. More than 75% of 7 alpha (OH)C is carried in blood by the LDLs.

Synthesis of a large peptide mimicking the DNA binding properties of the sex determining protein, SRY.

The sex determining protein, SRY, has been recently described as containing a DNA binding motif, also called the SRY box. This 80 amino acid box was synthesized using the continuous flow solid-phase technique. The product was then purified and tested according to such diverse criteria as its intrinsic structure or its biological activity (DNA binding capacity), and compared to the full-length protein. The data indicate that the peptide is relevant for the properties described so far for the protein.

[Human testicular determining factor: from the gene to protein]. / Le facteur de détermination testiculaire humain: du gène à la protéine.

Since its cloning in 1990, the human SRY gene has been formally identified with the testis determining factor. The SRY gene encodes a 204 amino acid protein of the High Mobility Group family. Its ability to bind DNA, to bend DNA or to be translocated into the nuclear compartment of the cell have now been established. However, neither its mode of action nor the description of target genes have been described so far, and are the topics of many studies.

Lack of complete 1p19q deletion in a consecutive series of 12 WHO grade II gliomas involving the insula: a marker of worse prognosis?

1p19q codeletion in WHO grade II glioma (GIIG) is claimed to be a marker of better outcome and chemosensitivity. Through the molecular study of 12 insular GIIG, our goal was to investigate a possible anatomo-molecular relationship in insula, specific brain sub-region, known to be involved with high frequency in GIIG. Loss of heterozygosity on 1p and 19q chromosomes was assessed in all tumors. None complete deletion of the 1p and 19q arms chromosomes and partial deletions in 3 patients were retrieved in the 11 WHO grade II oligodendrogliomas and one oligo-astrocytoma analyzed. Discrepancy between our results and the high reported incidence of 1p19q codeletion in oligodendrogliomas could mean that insular GIIG has a specific molecular pattern. We hypothesize that prognosis of insular GIIG is worse than in other locations, with reduced chemosensitivity. Thanks to minimization of surgical risks, surgery might be a first intention therapeutic option proposed in the GIIG.

Description and functional implications of a novel mutation in the sex-determining gene SRY.

The sex-determining gene SRY was screened for molecular alteration in an XY sex-reversed female by single-strand conformation polymorphism (SSCP) technique. An A-to-G transition was detected which leads to an exchange of a tyrosine by a cysteine in the SRY protein. The affected tyrosine residue located at the C terminus of the DNA binding protein is evolutionarily strongly conserved among the members of the HMG box containing proteins. Using gel shift assay and peptide synthesis such a mutation is shown to abolish the SRY protein DNA binding ability. The involvement of this particular amino acid in the binding specificity is also discussed.

Increase in free radicals on UHMWPE hip prostheses components due to inflamed synovial cell products.

Oxidative degradation of artificial UHMWPE joint implants caused by gamma-ray sterilization is thought to be responsible for the production of wear debris resulting in adverse tissue responses. On the other hand, it is well known that inflammation is associated with generation, by inflammatory cells, of free radicals (H(2)O(2) and NO) and destructive proteolytic enzymes (collagenases), which creates a strong oxidative environment. We hypothesized that when an UHMWPE implantation was performed in an inflammatory joint environment, the oxidative substances produced by inflamed synoviocytes could increase oxidative degradation of the polyethylene insert. We measured the amount of free radicals on conventional and on Duration-treated polyethylene samples by the electron spin resonance (ESR) technique before and after exposure of the samples to (1) inflamed synovial cell cultures; (2) normal synovial cell cultures; and (3) medium alone. We observed an increase in the number of free radicals on polyethylene samples after their immersion in cell cultures. Furthermore, it was observed that the increase of free radicals on polyethylene correlated with the degree of inflammation of synovial cells in culture.

SOX22 is a new member of the SOX gene family, mainly expressed in human nervous tissue.

SOX (SRY box-containing) genes share a particular DNA-binding domain, called HMG, with the mammalian testis-determining gene SRY Several SOX genes have already been shown to be transcription factors involved in the decision of important cell fates during development. Here we report the cloning of a new human member of the SOX gene family, SOX22. The corresponding protein contains several domains that are also present in other paralogous SOX proteins. The SOX22 gene maps to chromosome 20 on band p13 and does not appear to be clustered with any other SOX gene mapped to date. SOX22 mRNA is expressed in various fetal and adult organs and tissues, suggesting that this gene plays roles in both differentiation and maintenance of several cell types.

The human SOX11 gene: cloning, chromosomal assignment and tissue expression.

The mammalian testis determining gene SRY contains an HMG box-related DNA binding motif. By analogy a family of genes related to SRY in the HMG domain have been called SOX (SRY box-related genes). We have cloned and characterized the human SOX11 gene using the partial cloning of both human and mouse SOX11 genes and mapped it to chromosome 1p25. The SOX11 sequence is strongly conserved with the chicken homologue and is related to SOX4. It contains several putative transcriptional either activator or repressor domains. SOX11 expression pattern is consistent with the hypothesis that this gene is important in the developing nervous system.