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Biomedical subjects

L Genestier

Publications and source records attributed to L Genestier.

26 records · Page 2Linked to original sources
PubMed1995

Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis.

Programmed cell death or apoptosis is characterized by typical morphological alterations. By transmission electron microscopy, apoptotic cells are identified by condensation of the chromatin in tight apposition to the nuclear envelope, alteration of the nuclear envelope and fragmentation of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display an early desintegration of cytoplasmic membrane and swelling of mitochondria. In this study we assessed by flow cytometry the sequential alterations of forward angle light scatter, 90 degrees light scatter, and fluorescence associated with fluorescein diacetate, rhodamine 123, and propidium iodide in two human B cell lines undergoing apoptosis induced by the topoisomerase II inhibitor VP-16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by sodium azide. At the same time intervals, cells were examined by transmission electron microscopy and by UV microscopy after staining with Hoechst 33342. We report that sequential changes in light scatters and fluorescein diacetate are similar in cells undergoing apoptosis or necrosis, whereas apoptosis is characterized by a slightly delayed decrease of mitochondrial activity as assessed by rhodamine 123 staining. Surprisingly a part of cells undergoing apoptosis displayed an early uptake of propidium iodide followed by a condensation and then a fragmentation of their nuclei. It is concluded that uptake of propidium iodide is a very early marker of cell death which does not discriminate between necrosis and apoptosis. Along with biochemical criteria, nuclear morphology revealed by staining with Hoechst 33342 would seem to be of the most simple and most discriminative assay of apoptosis.

Animals↗
PubMed1995

Apoptosis without decrease of cell DNA content.

Apoptosis of human B cells and murine T and B cells was analyzed by DNA agarose gel electrophoresis, clamped homogeneous electric field, measurement of cell DNA content by flow cytometry, transmission electron microscopy and by UV microscopy. Apoptosis was induced by etoposide (an inhibitor of topoisomerase II), by the calcium ionophore ionomycin or by cross-linking of membrane immunoglobulins (Ig) with anti-Ig-antibodies. Two types of apoptosis could be defined. Apoptosis resulting in small DNA fragments (180-200 base pairs and multiples thereof) was associated with a typical 'ladder' in agarose gel electrophoresis and a decrease in cell DNA content assessed by flow cytometry. Conversely apoptosis with large DNA fragments (100-150 kilobase pairs) was only demonstrated by clamped homogeneous electric field but was not associated with decreased cell DNA content or the observation of DNA ladders. Nuclear condensation without fragmentation was more frequent when apoptosis generated large DNA fragments. The type of apoptosis appears to be an intrinsic property of each cell type.

Apoptosis↗
PubMed1995

Tumor necrosis factor-alpha up-regulates Bcl-2 expression and decreases calcium-dependent apoptosis in human B cell lines.

Group I and Epstein-Barr virus-negative Burkitt's lymphoma cell lines and the B104 lymphoma cell line which expresses a phenotype of immature B cells undergo apoptosis after cross-linking of their surface Ig receptors or after exposure to a calcium ionophore. We show here that tumor necrosis factor (TNF)-alpha protects these B cell lines against Ca(2+)-dependent apoptosis. Protection was associated with up-regulation of bcl-2 mRNA and protein expression. The increase of Bcl-2 expression induced by TNF-alpha was inhibited by chelerythrine, a specific inhibitor of protein kinase C (PKC), suggesting that Bcl-2 expression was dependent on PKC activation. Furthermore, we show that phorbol esters and cyclosporin A (CsA), which prevent Ca(2+)-dependent apoptosis, up-regulated Bcl-2 expression. The effect of CsA on Bcl-2 expression is controlled by calcineurin since we have shown that FK506 but not rapamycin had the same effect on Bcl-2 expression, whereas okadaic acid, an inhibitor of phosphatases 1, 2A and 2C, was ineffective. These data provide direct evidence that TNF-alpha prevents Ca(2+)-dependent apoptosis by a Bcl-2-dependent mechanism mediated by PKC.

Apoptosis↗
PubMed1994

Apoptosis induced by polyclonal antilymphocyte globulins in human B-cell lines.

Antilymphocyte and antithymocyte globulins (ALG) are currently used as immunosuppressive agents in clinical transplantation and for the treatment of severe aplastic anemia. ALG contain a mixture of antibodies that recognize T- and B-cell-specific antigens but mostly nonlineage-specific molecules. We reported previously that ALG could inhibit the proliferation of activated B cells and B cell lines (Bonnefoy-Bérard et al, Blood 79:2164, 1992). We show here that ALG induce apoptosis of several human hematopoietic cell lines, as shown by nuclear condensation and fragmentation in fluorescence and electronic microscopy and by double-strand DNA breaks shown by DNA electrophoresis. Apoptosis was achieved without elevation of intracellular Ca2+ and requirement for mRNA and protein synthesis. Most of the B-cell lines tested (Epstein-Barr virus [EBV]-transformed lymphoblastoid cell lines, EBV-negative and groups I/III EBV-positive Burkitt's lymphoma cell lines, as well as other B-lymphoma cell lines) were susceptible to ALG-induced cytotoxicity. Myelomonocytic and T-cell lines were much less susceptible than B-cell lines. Susceptibility to ALG-induced cytotoxicity was not correlated with intracellular Bcl-2 level. Most cell lines that express high levels of Fas/Apo-1 antigen were susceptible to ALG. However, several lines of evidence support the conclusion that, in addition to Fas/Apo-1, other cell surface molecules can mediate ALG-induced apoptosis. The cytotoxic activity could be fully removed by adsorption on susceptible cell lines but not on a resistant cell line, indicating that it was mediated by antibodies specific for surface antigens expressed only on susceptible cell lines. Apoptosis was triggered by ALG F(ab')2 fragments as well as by intact ALG. This cytotoxic property of ALG may account for their antiproliferative effect and might contribute to some extent to the relatively lower risk of posttransplant lymphoproliferative disorders previously reported in ALG-treated patients.

Antilymphocyte Serum↗
PubMed1994

The phosphoprotein phosphatase calcineurin controls calcium-dependent apoptosis in B cell lines.

Group I Burkitt's lymphoma cell lines and the B104 lymphoma cell line which expresses a phenotype of immature B cells undergo apoptosis after cross-linking of their surface immunoglobulin (Ig) receptors or after exposure to a calcium ionophore, while protein kinase C (PKC)-activating phorbol esters prevent such apoptosis. We show here that blockade of the phosphoprotein phosphatase calcineurin or phosphatase 2B by cyclosporin A (CsA) also protects these B cell lines against Ca(2+)-dependent apoptosis but not against apoptosis triggered by the PKC inhibitor chelerythrine or by serum deprivation. Okadaic acid, an inhibitor of phosphatases 1, 2A and 2C was ineffective. Among a series of human cytokines tested, only interferon-alpha and tumor necrosis factor-alpha were shown to protect against Ca(2+)-dependent apoptosis when used alone or in combination with CsA. In contrast to phorbol esters which block the progression into the S/G2 phases of the cell cycle, CsA partially restored the proliferation of cells exposed to the calcium ionophore. Altogether these data provide indirect evidence for the control of B cell apoptosis by the serine/threonine phosphorylation status of yet undefined key cellular substrates.

Antibodies, Anti-Idiotypic↗
PubMed1994

Cyclosporin A and FK506 inhibit activation-induced cell death in the murine WEHI-231 B cell line.

The WEHI-231 B lymphoma cell line expresses the phenotype of immature B cells. Cross-linking of surface IgM induces programmed cell death (PCD) with typical features of apoptosis demonstrated by the decrease of cell DNA content, chromatin condensation, and nuclear fragmentation. Activation of protein kinase C (PKC) by phorbol esters was reported to protect WEHI-231 cells against apoptosis induced by ligation of antigen receptors. It was therefore hypothesized that PCD could result from a defect in PKC response with an imbalance in the phosphoinositide pathway in favor of Ca2+ mobilization. In support of this hypothesis, we show here that apoptosis can be readily triggered by the calcium ionophore ionomycin. Furthermore, pretreatment of cells with cyclosporin A or FK506 which inhibit selectively the phosphoprotein calcineurin, a calcium-and calmodulin-dependent serine/threonine phosphatase, protects WEHI-231 cells against apoptosis induced by ionomycin or ligation of surface IgM. Unlike phorbol esters, cyclosporin A did not impair the rise of intracellular Ca2+ induced by cross-linking of antigen receptors. Altogether, the data indicate that the phosphorylation status of yet undefined key cellular substrates controls the cellular response to calcium-dependent apoptotic signals in this B cell lymphoma.

Animals↗