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Sylvie Huet

Publications and source records attributed to Sylvie Huet.

7 recordsLinked to original sources
PubMed2006

Statistics for proteomics: experimental design and 2-DE differential analysis.

Proteomics relies on the separation of complex protein mixtures using bidimensional electrophoresis. This approach is largely used to detect the expression variations of proteins prepared from two or more samples. Recently, attention was drawn on the reliability of the results published in literature. Among the critical points identified were experimental design, differential analysis and the problem of missing data, all problems where statistics can be of help. Using examples and terms understandable by biologists, we describe how a collaboration between biologists and statisticians can improve reliability of results and confidence in conclusions.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

Mitochondrial localization and activity of P-glycoprotein in doxorubicin-resistant K562 cells.

It is now well-established that P-glycoprotein 170 (P-gp), an efflux pump involved in multidrug resistance (MDR) is overexpressed at the plasma membrane of doxorubicin-resistant K562 leukemia cells. Nevertheless, several results suggested: (i) that P-gp-mediated drug efflux was not the only mechanism involved in resistance; (ii) that intracellular compartments could accumulate the drug, preventing it from reaching its nuclear targets; (iii) that agents able to reverse multidrug resistance may lead to intracellular drug redistribution. We have studied the localization of P-gp in mitochondria as well as its functional properties in this compartment. Using several monoclonal antibodies (MoAbs) directed against different P-gp epitopes, a protein was detected in the cytoplasm of two doxorubicin-resistant K562 sublines and, by confocal laser scanning microscopy, this protein was shown to co-localize in the Golgi apparatus and in mitochondria, in equivalent proportions. Purified mitochondria were isolated from K562 cell variants; the presence of a protein of about 170 kDa and reacting with several anti-P-gp antibodies was assessed in MDR cells by Western blotting and flow cytometry. Functional assays have shown that mitochondrial P-gp was involved in doxorubicin accumulation inside the organelle but not in its efflux, suggesting an orientation of P-gp in the mitochondrial membrane inverse to that observed in the plasma membrane. A potential role for mitochondrial P-gp in MDR cells would be to protect the nucleus from doxorubicin. This is the first demonstration of the presence and functional activity of P-gp in mitochondria of MDR cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Major improvement of the reference method of the French drug resistance network for P-glycoprotein detection in human haematological malignancies.

The aim of this study was to improve significantly the sensitivity and specificity of the flow cytometric assay of P-glycoprotein (Pgp) implemented and validated by the laboratories of the French Drug Resistance Network [Huet S, Marie JP, Gualde N, Robert J. Reference method for detection of Pgp mediated multidrug resistance in human hematological malignancies: a method validated by the laboratories of the French Drug Resistance Network. Cytometry 1998;34:248-56] in cells displaying low level of resistance. Fluoresceine-conjugated monoclonal antibodies (Mabs) and propidium iodide were respectively replaced by phycoerythrin-conjugated Mabs and Sytox green. The removal of erythrocytes and granulocytes by density gradient was replaced by the lysis of erythrocytes after Mab incubation. Using these conditions, Pgp could be detected in the K-H30 line, which was negative in former studies, with Mab/Control ratios increasing by 3.7- to 5.9-fold, and Mab/Control ratios in the parental sensitive K562 line still ranging between 0.8 and 1.2. When tested on 16 blood samples from patients presenting haematological malignancies, six samples presented low positivity, which was not detected with the former method, while 10 samples remained negative with the two methods. Pgp was specifically detected in pathological blood cells in the six positive samples.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

PrBn, a major gene controlling homeologous pairing in oilseed rape (Brassica napus) haploids.

Precise control of chromosome pairing is vital for conferring meiotic, and hence reproductive, stability in sexually reproducing polyploids. Apart from the Ph1 locus of wheat that suppresses homeologous pairing, little is known about the activity of genes that contribute to the cytological diploidization of allopolyploids. In oilseed rape (Brassica napus) haploids, the amount of chromosome pairing at metaphase I (MI) of meiosis varies depending on the varieties the haploids originate from. In this study, we combined a segregation analysis with a maximum-likelihood approach to demonstrate that this variation is genetically based and controlled mainly by a gene with a major effect. A total of 244 haploids were produced from F(1) hybrids between a high- and a low-pairing variety (at the haploid stage) and their meiotic behavior at MI was characterized. Likelihood-ratio statistics were used to demonstrate that the distribution of the number of univalents among these haploids was consistent with the segregation of a diallelic major gene, presumably in a background of polygenic variation. Our observations suggest that this gene, named PrBn, is different from Ph1 and could thus provide complementary information on the meiotic stabilization of chromosome pairing in allopolyploid species.

Alleles↗
PubMed2003

Potassium channel expression level is dependent on the proliferation state in the GH3 pituitary cell line.

Previously, we showed that the peak density of the transient outward K(+) current (I(to)) expressed in GH3 cells was different in the S phase than in other phases of the cell cycle. Using cell synchronization, we show here that I(to) drops precisely at the quiescent (G(0) phase)/proliferating transition. This change is not due to a modification in the voltage dependence of I(to), but rather to a modification in its inactivation kinetics. Molecular determination of K(+) channel subunits showed that I(to) required the expression of Kv1.4, Kv4.1, and Kv4.3. We found that the increase in I(to) density during the quiescent state was accompanied by an increase in Kv1.4 protein expression, whereas Kv4.3 expression remained unchanged. We further demonstrate that the link between I(to) expression and cell proliferation is not mediated by variations in cell excitability. These results provide new evidence for the cell cycle dependence of I(to) expression, which could be relevant in understanding the mechanisms leading to pituitary adenomas.

Animals↗
PubMedDate not supplied

Quantification of the expression of multidrug resistance-related genes in human tumour cell lines grown with free doxorubicin or doxorubicin encapsulated in polyisohexylcyanoacrylate nanospheres.

Doxorubicin (dox) encapsulated in polyisohexylcyanoacrylate nanospheres (PIHCA-dox) can circumvent P-glycoprotein-mediated multidrug resistance (MDR). In order to investigate whether this drug formulation is able to select MDR cells in culture in the same way as free doxorubicin does, two human tumour cell lines, K562 and MCF7, were grown with increasing concentrations of either free dox or PIHCA-dox. For both drug formulations and for each selection level, the cell lines were more resistant to free dox than to PIHCA-dox. The MCF7 sublines selected with PIHCA-dox exhibited a higher level of resistance to both doxorubicin formulations than those selected with free doxorubicin. Different levels of overexpression of several genes involved in drug resistance (MDR1, MRP1, BCRP and TOP2alpha) occurred in the resistant variants. MDR1 gene overexpression was consistently higher in free dox-selected cells than in PIHCA-dox-selected cells, while this was the reverse for the BCRP gene. Overexpression of the MRP1 and TOP2alpha genes was also observed in the selected variants. Our results show that several mechanisms may be involved in the acquisition of drug resistance and that drug encapsulation markedly alters or delays these processes.

ATP Binding Cassette Transporter, Subfamily G, Mem↗