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Accueil > Agenda > Les séminaires Jean Roche > Imagerie de fluorescence du petit animal : limites, applications et (...)

Imagerie de fluorescence du petit animal : limites, (...)

Lundi 22 mai 2006,11h, salle Lissitzky.

Bibliographie

1 : Cancer Res. 2005 Dec 15 ;65(24):11667-75.

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

Pchejetski D, Golzio M, Bonhoure E, Calvet C, Doumerc N, Garcia V, Mazerolles C, Rischmann P, Teissie J, Malavaud B, Cuvillier O.

Inserm, U466, Toulouse, France.

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.


2 : Biochim Biophys Acta. 2005 Aug 5 ;1724(3):248-54. Epub 2005 Apr 21.

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

Phez E, Faurie C, Golzio M, Teissie J, Rols MP.

Institut de Pharmacologie et de Biologie Structurale du CNRS (UMR5089), 205, Route de Narbonne, 31077 Toulouse cedex 4, France.

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.


3 : Gene Ther. 2005 Feb ;12(3):246-51.

Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery.

Golzio M, Mazzolini L, Moller P, Rols MP, Teissie J.

IPBS CNRS (UMR 5089), Toulouse, France.

Owing to their capacity to induce strong, sequence-specific, gene silencing in cells, short interfering RNAs (siRNAs) represent new potential therapeutic tools. This development requires, however, new safe and efficient in vivo siRNA delivery methods. In the present technical report, we show that electrically mediated siRNA transfer can suppress transgene expression in adult mice muscles. Using electropulsation for siRNA delivery opens the way for a targeted gene silencing on a broad range of tissues. Clinical applications of electropulsation for delivery of other classes of molecules are under trials. We reported that gene silencing was efficiently obtained in vivo in an adult mammal (mouse) with chemically synthesized siRNA after its electrical delivery. The associated gene silencing was followed on the same animal and lasted at least 11 days. Gene silencing was obtained in muscles not only on young adult mice but also on much older animals. No tissue damages were detected under our electrical conditions. Therefore, this method should provide an efficient approach for a localized delivery of siRNAs in various tissues and organs.


4 : Biochim Biophys Acta. 2004 Oct 11 ;1665(1-2):92-100.

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

Faurie C, Phez E, Golzio M, Vossen C, Lesbordes JC, Delteil C, Teissie J, Rols MP.

Institut de Pharmacologie et de Biologie Structurale du CNRS UMR 5089, 205, route de Narbonne, 31077 Toulouse cedex, France.

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.


5 : Gene Ther. 2004 Oct ;11 Suppl 1:S85-91.

Optical imaging of in vivo gene expression : a critical assessment of the methodology and associated technologies.

Golzio M, Rols MP, Gabriel B, Teissie J.

IPBS/CNRS (UMR 5089), Toulouse, France.

Following and quantifying the expression of reporter gene expression in vivo is very important to monitor the expression of therapeutic genes in targeted tissues in disease models and/or to assess the effectiveness of systems of gene therapy delivery. Gene expression of luminescent or fluorescent proteins can be detected directly on living animals by simply observing the associated optical signals by means of a cooled charged-coupled device camera. More accurate resolution can be obtained with more sophisticated technologies. Time-course and quasi-quantitative monitoring of the expression can be obtained on a given animal and followed on a large time window. The present paper describes the physical and technological methodologies and associated problems of in vivo optical imaging. Several examples of in vivo detection of gene delivery are described.


6 : Methods. 2004 Jun ;33(2):126-35.

In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression.

Golzio M, Rols MP, Teissie J.

IPBS UMR 5089 CNRS, 205 route de Narbonne, 31077 Toulouse, France.

Electropulsation is one of the non-viral methods successfully used to transfer genes into living cells in vitro as in vivo. This approach shows promise in the field of gene and cellular therapies. The present paper first describes the factors controlling electropermeabilization to small molecules (< 4 kDa) and then the processes supporting DNA transfer in vitro. The description of in vitro events brings the attention of the reader to the processes occurring before, during, and after electropulsation of DNA and cells. Their developments for the in vivo processes are reported in the final part where the present and potential clinical applications are described. Copyright 2003 Elsevier Inc.


7 : J Soc Biol. 2003 ;197(3):301-10. [Calcium and electropermeabilized cells]

[Article in French]

Golzio M, Gabriel B, Boissier F, Deuwille J, Rols MP, Teissie J.

IPBS CNRS (UMR 5089) 205, route de Narbonne, 31077 Toulouse.

Trains of short and intense electric pulses may induce a reversible local permeabilization on the membrane of the treated cells. Hydrophilic species can then almost freely cross the envelope and either enter or escape from the cytoplasm. The purpose of the present study was to investigate the possibility of introducing well defined amounts of Ca2+ ions within the cell. Chinese hamster ovary cells were used as a model system. When the pulsing buffer contained high levels of free Ca2+, the survival of cells was strongly affected. A 1 mM level was well tolerated. When cells were pulsed under moderated field conditions, it was observed that Ca2+ entered cells very rapidly (second time range). But the basic cytoplasmic level was set back spontaneously within a few minutes. The perspectives of this electrical injection are discussed for basic cell biology and high-throughput biotechnology.


8 : DNA Cell Biol. 2003 Dec ;22(12):777-83.

Cell and animal imaging of electrically mediated gene transfer.

Faurie C, Golzio M, Moller P, Teissie J, Rols MP.

Institut de Pharmacologie et de Biologie Structurale du CNRS, Toulouse, France.

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.


9 : Technol Cancer Res Treat. 2002 Oct ;1(5):319-28.

Factors controlling electropermeabilisation of cell membranes.

Rols MP, Golzio M, Gabriel B, Teissie J.

IPBS UMR 5089 CNRS, 205 route de Narbonne, 31077 Toulouse, France.

Electric field pulses are a new approach for drug and gene delivery for cancer therapy. They induce a localized structural alteration of cell membranes. The associated physical mechanisms are well explained and can be safely controlled. A position dependent modulation of the membrane potential difference is induced when an electric field is applied to a cell. Electric field pulses with an overcritical intensity evoke a local membrane alteration. A free exchange of hydrophilic low molecular weight molecules takes place across the membrane. A leakage of cytosolic metabolites and a loading of polar drugs into the cytoplasm are obtained. The fraction of the cell surface which is competent for exchange is a function of the field intensity. The level of local exchange is strongly controlled by the pulse duration and the number of successive pulses. The permeabilised state is long lived. Its lifetime is under the control of the cumulated pulse duration. Cell viability can be preserved. Gene transfer is obtained but its mechanism is not a free diffusion. Plasmids are electrophoretically accumulated against the permeabilised cell surface and form aggregates due to the field effect. After the pulses, several steps follow : translocation to the cytoplasm, traffic to the nucleus and expression. Molecular structural and metabolic changes in cells remain mostly poorly understood. Nevertheless, while most studies were established on cells in culture (in vitro), recent experiments show that similar effects are obtained on tissue (in vivo). Transfer remains controlled by the physical parameters of the electrical treatment.


10 : Biochim Biophys Acta. 2002 Jun 13 ;1563(1-2):23-8.

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

Golzio M, Teissie J, Rols MP.

Institut de Pharmacologie et de Biologie Structurale du CNRS UMR 5089, 205, route de Narbonne, 31077 Cedex Toulouse, France.

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.


11 : Proc Natl Acad Sci U S A. 2002 Feb 5 ;99(3):1292-7. Epub 2002 Jan 29.

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

Golzio M, Teissie J, Rols MP.

Institut de Pharmacologie et de Biologie Structurale Centre National de la Recherche Scientifique/Unite Mixte de Recherche-5089, 205, Route de Narbonne, 31077 Toulouse Cedex, France.

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.

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