Biomedical subjects

Muriel Golzio

Publications and source records attributed to Muriel Golzio.

9 recordsLinked to original sources

PubMed2006

New anti angiogenesis developments through electro-immunization: optimization by in vivo optical imaging of intradermal electro gene transfer.

Direct application of high voltage electric pulses of milliseconds duration to the skin of a mouse enhances in vivo intradermal delivery of injected therapeutic molecules such as DNA. The efficacy of gene transfer and expression is dependent on electrical parameters. DNA electrotransfer in tissues increases the associated DNA expression vaccine potency. This protocol is called "electro-immunization". In the present study, we report a new strategy for optimizing electro-immunization. In vivo fluorescence imaging was used to detect the expression of a fluorescent protein (DsRed) and therefore allowed rapid optimization of the protocol. In vivo electrogenetransfer in the skin was well tolerated and DsRed expression was followed for over 2 weeks. Expression was voltage dependent under our conditions. Parameters were selected giving the highest level of expression. Under these optimized conditions, electrotransfer of a plasmid encoding VEGF was evaluated for its immune response as a gene therapy of interest involved in anti-angiogenic strategies. Anti VEGF 165 antibodies in sera of mice were evaluated by ELISA and compared to those obtained after conventional immunization. Comparable titres of antibodies were obtained in both groups. An IgG2a predominance was found in mice immunized with the plasmid whereas a IgG1 predominance was observed in mice immunized classically. Skin electro-immunization is therefore shown as a good route for DNA immunization for anti-angiogenesis concern.

Animals↗

PubMed2006

In vivo imaging of tumor growth after electrochemotherapy with cisplatin.

Imaging methods can give both temporal and spatial dimensions to characterize the processes in progression of and/or treatment of specific disease Subcutaneous tumors can be cured after electrochemotherapy (ECT). Growth and reduction of tumors as a result of cytotoxic therapy can be followed by fluorescence video imaging directly on the same animal after treatment. Imaging of tumors should bring more information on the cellular effects of ECT. Green fluorescent protein (eGFP) expressing B16F10 and LPB tumors implanted in C57Bl/6 mice were treated with ECT with cisplatin. The growth or regression of the tumors was monitored either classically by using a caliper or by a manual definition of the region of interest where critical fluorescence levels were detected on the animals. A very good correlation between the two methods was observed. The eGFP mean fluorescence emission was only slightly affected by ECT with intravenously injected cisplatin. Ex vivo observations under a fluorescence microscope showed that eGFP was only detected on the outer layer of the tumor. No fluorescence was detected in the central part of the tumors, which were necrotic.

Animals↗

PubMed2005

Sphingosine kinase-1 as a chemotherapy sensor in prostate adenocarcinoma cell and mouse models.

Systemic chemotherapy was considered of modest efficacy in prostate cancer until the recent introduction of taxanes. We took advantage of the known differential effect of camptothecin and docetaxel on human PC-3 and LNCaP prostate cancer cells to determine their effect on sphingosine kinase-1 (SphK1) activity and subsequent ceramide/sphingosine 1-phosphate (S1P) balance in relation with cell survival. In vitro, docetaxel and camptothecin induced strong inhibition of SphK1 and elevation of the ceramide/S1P ratio only in cell lines sensitive to these drugs. SphK1 overexpression in both cell lines impaired the efficacy of chemotherapy by decreasing the ceramide/S1P ratio. Alternatively, silencing SphK1 by RNA interference or pharmacologic inhibition induced apoptosis coupled with ceramide elevation and loss of S1P. The differential effect of both chemotherapeutics was confirmed in an orthotopic PC-3/green fluorescent protein model established in nude mice. Docetaxel induced a stronger SphK1 inhibition and ceramide/S1P ratio elevation than camptothecin. This was accompanied by a smaller tumor volume and the reduced occurrence and number of metastases. SphK1-overexpressing PC-3 cells implanted in animals developed remarkably larger tumors and resistance to docetaxel treatment. These results provide the first in vivo demonstration of SphK1 as a sensor of chemotherapy.

Adenocarcinoma↗

PubMed2005

New insights in the visualization of membrane permeabilization and DNA/membrane interaction of cells submitted to electric pulses.

Electropermeabilization designates the use of electric pulses to overcome the barrier of the cell membrane. This physical method is used to transfer anticancer drugs or genes into living cells. Its mechanism remains to be elucidated. A position-dependent modulation of the membrane potential difference is induced, leading to a transient and reversible local membrane alteration. Electropermeabilization allows a fast exchange of small hydrophilic molecules across the membrane. It occurs at the positions of the cell facing the two electrodes on an asymmetrical way. In the case of DNA transfer, a complex process is present, involving a key step of electrophoretically driven association of DNA only with the destabilized membrane facing the cathode. We report here at the membrane level, by using fluorescence microscopy, the visualization of the effect of the polarity and the orientation of electric pulses on membrane permeabilization and gene transfer. Membrane permeabilization depends on electric field orientation. Moreover, at a given electric field orientation, it becomes symmetrical for pulses of reversed polarities. The area of cell membrane where DNA interacts is increased by applying electric pulses with different orientations and polarities, leading to an increase in gene expression. Interestingly, under reversed polarity conditions, part of the DNA associated with the membrane can be removed, showing some evidence for two states of DNA in interaction with the membrane: DNA reversibly associated and DNA irreversibly inserted.

Animals↗

PubMed2004

Effect of electric field vectoriality on electrically mediated gene delivery in mammalian cells.

Electropermeabilization is a nonviral method used to transfer genes into living cells. Up to now, the mechanism is still to be elucidated. Since cell permeabilization, a prerequired for gene transfection, is triggerred by electric field, its characteristics should depend on its vectorial properties. The present investigation addresses the effect of pulse polarity and orientation on membrane permeabilization and gene delivery by electric pulses applied to cultured mammalian cells. This has been directly observed at the single-cell level by using digitized fluorescence microscopy. While cell permeabilization is only slightly affected by reversing the polarity of the electric pulses or by changing the orientation of pulses, transfection level increases are observed. These last effects are due to an increase in the cell membrane area where DNA interacts. Fluorescently labelled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized cell surface is stable and is not affected by pulses of reversed polarities. Under such conditions, DNA interacts with the two sites of the cell facing the two electrodes. When changing both the pulse polarity and their direction, DNA interacts with the whole membrane cell surface. This is associated with a huge increase in gene expression. This present study demonstrates the relationship between the DNA/membrane surface interaction and the gene transfer efficiency, and it allows to define the experimental conditions to optimize the yield of transfection of mammalian cells.

Animals↗

PubMed2003

Effect of electric field induced transmembrane potential on spheroidal cells: theory and experiment.

The transmembrane potential on a cell exposed to an electric field is a critical parameter for successful cell permeabilization. In this study, the effect of cell shape and orientation on the induced transmembrane potential was analyzed. The transmembrane potential was calculated on prolate and oblate spheroidal cells for various orientations with respect to the electric field direction, both numerically and analytically. Changing the orientation of the cells decreases the induced transmembrane potential from its maximum value when the longest axis of the cell is parallel to the electric field, to its minimum value when the longest axis of the cell is perpendicular to the electric field. The dependency on orientation is more pronounced for elongated cells while it is negligible for spherical cells. The part of the cell membrane where a threshold transmembrane potential is exceeded represents the area of electropermeabilization, i.e. the membrane area through which the transport of molecules is established. Therefore the surface exposed to the transmembrane potential above the threshold value was calculated. The biological relevance of these theoretical results was confirmed with experimental results of the electropermeabilization of plated Chinese hamster ovary cells, which are elongated. Theoretical and experimental results show that permeabilization is not only a function of electric field intensity and cell size but also of cell shape and orientation.

Animals↗

PubMed2003

Cell and animal imaging of electrically mediated gene transfer.

Electropermeabilization is a nonviral method successfully used to transfer genes into cells in vitro as in vivo. Although it shows promise in field of gene therapy, very little is known on the basic processes supporting the DNA transfer. The aim of the present investigation is to visualize gene electrotransfer and expression both in vitro and in vivo. In vitro studies have been performed by using digitized fluorescence microscopy. Membrane permeabilization occurs at the sides of the cell membrane facing the two electrodes. A free diffusion of propidium iodide across the membrane to the cytoplasm is observed in the seconds following electric pulses. Fluorescently labeled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized cell surface is stable over a few minutes. Changing the polarity and the orientation of the pulses lead to an increase in gene expression. In vivo experiments have been performed in Tibialis Cranialis mice muscle. Electric field application lead to the in vivo expression of plasmid DNA. We directly visualize gene expression of the Green Fluorescent Protein (GFP) on live animals. GFP expression is shown to be increased by applying electric field pulses with different polarities and orientations.

Animals↗

PubMed2002

Cell synchronization effect on mammalian cell permeabilization and gene delivery by electric field.

Electropermeabilization is a promising nonviral method for gene therapy. However, despite the fact that it is widely used to transfer genes into living cells, the steps that limit DNA transfer remain to be determined. Here, we report the effect of cell synchronization on membrane permeabilization and gene delivery by electric fields. Chinese hamster ovary (CHO) cells were synchronized by aphidicolin or butyrate treatment. Electro-mediated transfection of these cells was evaluated under electric field conditions leading to the same level of membrane permeabilization. Aphidicolin cell synchronization in G2/M phase leads to a slight increase in plasma membrane permeabilization but to a three-fold increase in percentage of transfected cells and to an eight-fold increase in gene expression. This increase in cell transfection is specifically due to the G2/M synchronization process. Indeed, cell synchronization in G1 phase by sodium butyrate has no effect on cell permeabilization and transfection. Our results suggest that the enhanced transfection level in G2/M phase is not simply due to enhanced permeabilization, but reinforce the statement that the melting of the nuclear membrane facilitates direct access of plasmid DNA to the nucleus.

Animals↗

PubMed2002

Direct visualization at the single-cell level of electrically mediated gene delivery.

Electropermeabilization is one of the nonviral methods successfully used to transfer genes into living cells in vitro and in vivo. Although this approach shows promise in the field of gene therapy, very little is known about the basic processes supporting DNA transfer. The present investigation studies this process at the single-cell level by using digitized fluorescence microscopy. Permeabilization is a prerequisite for gene transfer. Its assay by propidium-iodide (PI) penetration shows that it occurs at the sides of the cell membrane facing the two electrodes, whereas fluorescently labeled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized part of the cell surface results in the formation of localized aggregates. These membrane-associated spots are formed only when pulses with a longer duration than a critical value are applied. These complexes are formed within 1 s after the pulses and cannot be destroyed by pulses of reversed polarities. They remain at the membrane level up to 10 min after pulsing. Although freely accessible to DNA dye (TOTO-1) 1 min after the pulses, they are fully protected when the addition takes place 10 min after. They diffuse in the cytoplasm 30 min after pulses and are present around the nucleus 24 h later.

Animals↗