Genome-wide microarray expression and genomic alterations by array-CGH analysis in neuroblastoma stem-like cells.

Neuroblastoma has a very diverse clinical behaviour: from spontaneous regression to a very aggressive malignant progression and resistance to chemotherapy. This heterogeneous clinical behaviour might be due to the existence of Cancer Stem Cells (CSC), a subpopulation within the tumor with stem-like cell properties: a significant proliferation capacity, a unique self-renewal capacity, and therefore, a higher ability to form new tumors. We enriched the CSC-like cell population content of two commercial neuroblastoma cell lines by the use of conditioned cell culture media for neurospheres, and compared genomic gains and losses and genome expression by array-CGH and microarray analysis, respectively (in CSC-like versus standard tumor cells culture). Despite the array-CGH did not show significant differences between standard and CSC-like in both analyzed cell lines, the microarray expression analysis highlighted some of the most relevant biological processes and molecular functions that might be responsible for the CSC-like phenotype. Some signalling pathways detected seem to be involved in self-renewal of normal tissues (Wnt, Notch, Hh and TGF-β) and contribute to CSC phenotype. We focused on the aberrant activation of TGF-β and Hh signalling pathways, confirming the inhibition of repressors of TGF-β pathway, as SMAD6 and SMAD7 by RT-qPCR. The analysis of the Sonic Hedgehog pathway showed overexpression of PTCH1, GLI1 and SMO. We found overexpression of CD133 and CD15 in SIMA neurospheres, confirming that this cell line was particularly enriched in stem-like cells. This work shows a cross-talk among different pathways in neuroblastoma and its importance in CSC-like cells

A constitutive BCL2 down-regulation aggravates the phenotype of PKD1-mutant-induced polycystic kidney disease

IF 5.340International audienceThe main identified function of BCL2 protein is to prevent cell death by apoptosis. Mice knock-out for Bcl2 demonstrate growth retardation, severe polycystic kidney disease (PKD), gray hair and lymphopenia, and die prematurely after birth. Here, we report a 40-year-old male referred to for abdominal and thoracic aortic dissection with associated aortic root aneurysm, PKD, lymphocytopenia with a history of T cell lymphoblastic lymphoma, white hair since the age of 20, and learning difficulties. PKD, which was also detected in the father and sister, was related to an inherited PKD1 mutation. The combination of PKD with gray hair and lymphocytopenia was also reminiscent of Bcl2-/- mouse phenotype. BCL2 gene transcript and protein level were observed to be dramatically decreased in patient peripheral blood T-cells and in his aorta vascular wall cells, which was not detected in parents and sister T-cells, suggesting an autosomal recessive inheritance. Accordingly, spontaneous apoptosis of patient T-cells was increased and could be rescued through stimulation with an anti-CD3 antibody. Direct sequencing of BCL2 gene exons, promoter and 3'UTR region as well as BCL2 mRNA sequencing failed in identifying any pathogenic variant. Array-CGH was also normal and whole exome sequencing of the patient, parents and sister DNA did not detect any significant variant in genes encoding Bcl2-interacting proteins. miRNA array identified an up-regulation of miR-181a, which is a known regulator of BCL2 expression. Altogether, miR-181a-mediated decrease in BCL2 gene expression could be a modifying factor that aggravates the phenotype of a PKD1 constitutive variant

Identification de biomarqueurs génétiques potentiels liés à l’apparition de pathologies cardiaques chez les enfants ayant survécu à un cancer : étude cas-témoins

National audienc

Identification of potential genetic biomarkers related to cardiac diseases occurrence in childhood cancer survivors: a case-control study.

International audienc

Whole chromosome plots.

<p>Array-CGH from SK-N-DZ (A) and SIMA (B) cell lines. The X-axis represents the chromosomes while the Y-axis represent the normalize log2 ratio fluorescence intensity thresholds −1 (loss) and 1 (gain). The results show gains and losses of small chromosomal regions.</p

RT-qPCR for Sonic Hedgehog (A) and TGF-β pathways analysis (B) in SIMA CSC-like cells.

<p>The graphs represent the 2^−ΔΔCt values obtained by RT-qPCR for neurospheres. The dotted line indicates the 2^−ΔΔCt control (cell line) value equal to 1. Significance against control: p<0.05 (*); p<0.01 (**); p<0.001 (***) and p<0.0001 (****). Selected genes cover the most relevant components of each pathway. Results confirmed aberrant activation of Hh (A) and TGF-β (B) signalling pathways in CSC-like cells.</p

MicroRNA identified in array-CGH analysis.

<p>MicroRNA identified in array-CGH analysis.</p

PANTHER classification by signalling pathway.

<p>The differentially expressed genes in both SK-N-DZ and SIMA CSC-like cells were classified by PANTHER and graphed. The percentage represents the number of genes altered against total number of genes involved in each pathway.</p

Gene expression by RT-qPCR.

<p>The graphs represent the 2^−ΔΔCt values obtained by RT-qPCR for neurospheres. The dotted line indicates the 2^−ΔΔCt control (cell line) value equal to 1. Significance against control: p<0.05 (*); p<0.01 (**); p<0.001 (***) and p<0.0001 (****). <b>Corroboration of expression array (A).</b> Both SK-N-DZ (▪) and SIMA (▴) CSC-like cells showed a similar expression profile. <b>Stem Cell Markers expression (B).</b> Results confirmed a high overexpression of CSC markers in SIMA cell line after the neurosphere formation assay.</p

Upregulated (UR, red) and downregulated (DR, green) genes (>1 fold-change) in neurospheres compared to standard cells lines SK-N-DZ and SIMA (A), represented with MAplots, were the Y-axis represents the red/green intensity ratio “M” and the X-axis the average intensity “A”.

<p>Venn diagrams illustrating the number of differentially expressed genes in SK-N-DZ and SIMA neurospheres compared to standard cell lines (B). The overlapping area represents the set of genes altered in both cell lines.</p