How transient alterations of organelles in mammalian cells submitted to electric field may explain some aspects of gene electrotransfer process.

Electric pulses can be used to transiently permeabilize the cell plasma membrane. This method is nowadays employed as a safe and efficient means to deliver therapeutic molecules into target cells and tissues. Despite the large bulk of literature on this topic, there is a lack of knowledge about the mechanism(s) of molecule delivery. The behavior of the cells both while the field is on and after its application is indeed not well described. Questions about cell organelle alterations remain unanswered. We report here evidence for a number of ultrastructural alterations in mammalian cells exposed to electric pulses. Specifically, CHO cells were subjected to trains of 10 pulses lasting 5ms using an electric field of 800V/cm, i.e. under conditions leading both to membrane permeabilization, gene transfer and expression. Cells were observed to undergo morphological alterations of the mitochondria and nucleus. These modifications, detected in the minutes following pulse delivery, were transient. They may have direct consequences on molecule delivery and therefore may explain various aspects of the mechanisms of DNA electrotransfer.

Cholesterol implications in plasmid DNA electrotransfer: Evidence for the involvement of endocytotic pathways.

The delivery of therapeutic molecules such as plasmid DNA in cells and tissues by means of electric fields holds great promise for anticancer treatment. To allow for their therapeutic action, the molecules have first to traverse the cell membrane. The mechanisms by which the electrotransferred pDNA interacts with and crosses the plasma membrane are not yet fully explained. The aim of this study is to unravel the role of cholesterol during gene electrotransfer in cells. We performed cholesterol depletion experiments and measured its effects on various steps of the electroporation process. The first two steps consisting of electropermeabilization of the plasma membrane and of pDNA interaction with it were not affected by cholesterol depletion. In contrast, gene expression decreased. Colocalization studies with endocytotic markers showed that pDNA is endocytosed with concomitant clathrin- and caveolin/raft-mediated endocytosis. Cholesterol might be involved in the pDNA translocation through the plasma membrane. This is the first direct experimental evidence of the occurrence of endocytosis in gene electrotransfer.

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.

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.

Polynorbornene polycationic polymers as gene transfer agents. Influence of the counterion for in vitro transfection.

Polycationic derivatives of polynorbornene with different non-cytotoxic counterions, have been prepared by organometallic polymerization of methyleneammonium norbornene and subsequent exchange of the counterion. In this paper the effect of the counterion on the polycationic polymer binding onto plasmid DNA was studied via different ethidium bromide assays, heparin displacement and protection against degradation by DNAse. According to the nature of the counterions and consequently the size of the polymer particles, their complexation with the DNA led to aggregates with variable binding affinity for the plasmid. The relative transfection efficiency of each polyplex was compared, on the basis of reporter gene expression, in cells in culture. The nature of the counterion was seen to affect gene delivery. The order of transfection efficiency of the counterions studied at equivalent charge ratios (NH3+/PO4-) is lactobionate, acetate, chloride. The results obtained with the polynorbornene methyleneammonium lactobionate and acetate are particularly encouraging.

Kinetic mechanism for the formation of the presynaptic complex of the bacterial recombinase RecA.

RecA protein from Escherichia coli catalyzes DNA strand exchange during homologous recombination in a reaction that requires nucleoside triphosphate cofactor. In the first step of this reaction RecA protein polymerizes on single-stranded DNA to form a filament with a stoichiometry of three nucleotides/RecA monomer called the presynaptic complex. We have used fluorescence anisotropy of a fluorescein-labeled oligonucleotide to investigate presynaptic complex formation. RecA-ATPgammaS bound to oligonucleotide by a two-step process. Kinetic studies revealed an intermediate in the polymerization reaction that had greater mobility than the final product filament. The intermediate was transformed into the final product by a process that was independent of filament concentration and temperature, k = 0.3 +/- 0.1 min(-1). This process had the same rate as that reported for a step in the isomerization of presynaptic complex by ATPgammaS (Paulus, B. F., and Bryant, F. R. (1997) Biochemistry 36, 7832-7838). Judging from anisotropy measurements, the intermediate had hydrodynamic properties similar to a mixed filament containing RecA monomers with and without ATPgammaS. These results show that the presynaptic complex can assume conformations with different segmental mobilities that could play a role in homologous recombination.