Biomedical subjects

Amandine Pitaval

Publications and source records attributed to Amandine Pitaval.

7 recordsLinked to original sources

PubMed2006

Cell microarrays in drug discovery.

There is an increasing need for systematic cell-based assays in a high-throughput screening (HTS) format to analyze the phenotypic consequences of perturbing mammalian cells with drugs, genes, interfering RNA. Taking advantage of the recent progress in microtechnology, new cell microarrays are being developed and applied to a large range of issues in metazoan cells. This article compares different approaches and evaluates their potential use in the drug discovery process. Although still an emerging technology, cell microarrays hold great promise to optimize the efficiency:cost ratio in cell-based HTS.

Animals↗

PubMed2005

Global transcriptional characterization of SP and MP cells from the myogenic C2C12 cell line: effect of FGF6.

With the use of Hoechst staining techniques, we have previously shown that the C2C12 myogenic cell line contains a side population (SP) that is largely increased in the presence of fibroblast growth factor 6 (FGF6). Here, we compared transcriptional profiles from SP and main population (MP) cells from either C2C12 or FGF6-expressing C2C12. Expression profiles of SPs show that these cells are less differentiated than MPs and display some similarities to stem cells. Moreover, principal component analysis made it possible to distinguish specific contributions of either FGF6 or differentiation effects on gene expression profiles. This demonstrated that FGF6-expanded SPs were similar to parental C2C12-derived SPs. Conversely, FGF6-treated MPs differed from parental MPs and were more related to SP cells. These results show that FGF6 pushed committed myogenic cells toward a more immature phenotype resulting in the accumulation of cells with a SP phenotype. We propose that FGF6 conditioning could provide a way to expand the pool of immature cells by myoblast dedifferentiation.

Animals↗

PubMed2005

Id2 reverses cell cycle arrest induced by {gamma}-irradiation in human HaCaT keratinocytes.

Id2 plays a key role in epithelial cells, regulating differentiation, the cell cycle, and proliferation. Because human skin constantly renews itself and is the first target of irradiation, it is of primary interest to evaluate whether such a gene may be regulated in keratinocytes exposed to ionizing radiation. We show here that Id2 is induced in response to gamma-irradiation and have investigated the consequence of this regulation on cell fate. Using RNA interference, we observed that Id2 extinction significantly reduces cell growth in human keratinocytes through the control of the G(1)-S transition of the cell cycle. We have investigated whether the impact of Id2 on the cell cycle may have a physiological role on the cell's ability to cope with radiative stress. Indeed, when Id2 is down-regulated through interfering RNA, cells are more sensitive to irradiation. Conversely, when Id2 is overexpressed, this somehow protects the cell. We propose that Id2 favors reentering the cell cycle after radiation-induced cell cycle arrest to permit the recovery of keratinocytes exposed to ionizing radiation.

Cell Cycle↗

PubMed2005

[Cell microarrays and functional genomics].

With the complete sequencing of the human genome, research priorities have shifted from the identification of genes to the elucidation of their function. Methods currently used by scientists to characterize gene function, such as knock-out mice, are based upon loss of protein function and analysis of the resulting phenotypes to infer a potential role for the protein under scrutiny. Until now, these methods have been successful but time consuming and only a few genes at a time could be analyzed. Cell microarrays allow to simultaneously transfect thousands of different nucleic acid molecules, RNA or DNA, into adherent cells. It is then possible to analyze a large pallet of resulting phenotypes in clusters of transfected cells. We are currently manufacturing cell microarrays with collections of full-length cDNA cloned in expression vectors (gain of function analyses) or siRNA (loss of function studies) to unravel function of genes involved in differentiation and proliferation of human cells. Although there are still some technological difficulties to overcome, the potential for cell microarrays to speed up functional exploration of genomes is very promising.

Cells, Cultured↗

PubMed2004

Expression profiling of genes and proteins in HaCaT keratinocytes: proliferating versus differentiated state.

The knowledge of the mechanism of keratinocyte differentiation in culture is still uncompleted. The emergence of new technologies, such as cDNA microarrays or 2D electrophoresis followed by mass spectrometry analysis, has allowed the identification of genes and proteins expressed in biological processes in keratinocytes. Here, we report a genome wide analysis of proliferating versus differentiated human HaCaT keratinocytes. We found that genes and proteins which take part in the cell cycle control, carbohydrate metabolism, cell auto-immunity, adhesion and cytokine signal transduction pathways were regulated in differentiated HaCaT keratinocytes. In addition, we identified seven proteins and 33 transcripts that had not been previously described as differentially expressed in proliferating versus differentiated HaCaT cells. Furthermore, some of these transcripts or proteins were similarly regulated in human primary keratinocytes and in human epidermis. The present study opens new areas of investigation in the comprehension of keratinocyte differentiation.

Animals↗

PubMed2004

Quantitative analysis of highly parallel transfection in cell microarrays.

As more genomes are sequenced, we are facing the challenge of rapidly unraveling the functions of genes. To that end, cell microarrays have recently been described that transfect thousands of nucleic acids in parallel and can be used to analyze the phenotypic consequences of such perturbations. As many parameters can influence the efficacy of transfection in such a format, we describe some important features in manufacturing cell microarrays that may improve reliability and efficiency of both plasmid DNA and siRNA transfection. We have also developed image analysis software that allows automatic detection of cell clusters, quantification of transfection efficiency and levels of expression/extinction of genes. Along with cell microarrays, this bioinformatic tool should expedite functional exploration of the human genome.

Automation↗

PubMed2004

Prion protein prevents human breast carcinoma cell line from tumor necrosis factor alpha-induced cell death.

To define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor alpha (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrP(c)), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining that indicated that PrP(c) is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adenoviruses expressing the human PrP(c,) our data demonstrate that PrP(c) overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation.

Breast Neoplasms↗