Christine Delteil

Coordinated assembly of Ku and p460 subunits of the DNA-dependent protein kinase on DNA ends is necessary for XRCC4-ligase IV recruitment.

Repair of DNA double-strand breaks by the non-homologous end-joining pathway (NHEJ) requires a minimal set of proteins including DNA-dependent protein kinase (DNA-PK), DNA-ligase IV and XRCC4 proteins. DNA-PK comprises Ku70/Ku80 heterodimer and the kinase subunit DNA-PKcs (p460). Here, by monitoring protein assembly from human nuclear cell extracts on DNA ends in vitro, we report that recruitment to DNA ends of the XRCC4-ligase IV complex responsible for the key ligation step is strictly dependent on the assembly of both the Ku and p460 components of DNA-PK to these ends. Based on co-immunoprecipitation experiments, we conclude that interactions of Ku and p460 with components of the XRCC4-ligase IV complex are mainly DNA-dependent. In addition, under p460 kinase permissive conditions, XRCC4 is detected at DNA ends in a phosphorylated form. This phosphorylation is DNA-PK-dependent. However, phosphorylation is dispensable for XRCC4-ligase IV loading to DNA ends since stable DNA-PK/XRCC4-ligase IV/DNA complexes are recovered in the presence of the kinase inhibitor wortmannin. These findings extend the current knowledge of the assembly of NHEJ repair proteins on DNA termini and substantiate the hypothesis of a scaffolding role of DNA-PK towards other components of the NHEJ DNA repair process.

Control by Osmotic Pressure of Voltage-Induced Permeabilization and Gene Transfer in Mammalian Cells

Cells can be transiently permeabilized by a membrane potential difference increase induced by the application of high electric pulses. This was shown to be under the control of the pulsing buffer osmotic pressure, when short pulses were applied. In this paper, the effects of buffer osmotic pressure during electric treatment and during the following 10 min were investigated in Chinese hamster ovary cells subjected to long (ms) square wave pulses, a condition needed to mediate gene transfer. No effect on cell permeabilization for a small molecule such as propidium iodide was observed. The use of a hypoosmolar buffer during pulsation allows more efficient loading of cells with beta-galactosidase, a tetrameric protein, but no effect of the postpulse buffer osmolarity was observed. The resulting expression of plasmid coding for beta-galactosidase was strongly controlled by buffer osmolarity during as well as after the pulse. The results, tentatively explained in terms of the effect of osmotic pressure on cell swelling, membrane organization, and interaction between molecules and membrane, support the existence of key steps in plasmid-membrane interaction in the mechanism of cell electrically mediated gene transfer.

Interplay between Ku, Artemis, and the DNA-dependent protein kinase catalytic subunit at DNA ends.

Repair of DNA double strand breaks (DSB) by the nonhomologous end-joining pathway in mammals requires at least seven proteins involved in a simplified two-step process: (i) recognition and synapsis of the DNA ends dependent on the DNA-dependent protein kinase (DNA-PK) formed by the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs in association with Artemis; (ii) ligation dependent on the DNA ligase IV·XRCC4·Cernunnos-XLF complex. The Artemis protein exhibits exonuclease and endonuclease activities that are believed to be involved in the processing of a subclass of DSB. Here, we have analyzed the interactions of Artemis and nonhomologous end-joining pathway proteins both in a context of human nuclear cell extracts and in cells. DSB-inducing agents specifically elicit the mobilization of Artemis to damaged chromatin together with DNA-PK and XRCC4/ligase IV proteins. DNA-PKcs is necessary for the loading of Artemis on damaged DNA and is the main kinase that phosphorylates Artemis in cells damaged with highly efficient DSB producers. Under kinase-preventive conditions, both in vitro and in cells, Ku-mediated assembly of DNA-PK on DNA ends is responsible for a dissociation of the DNA-PKcs·Artemis complex. Conversely, DNA-PKcs kinase activity prevents Artemis dissociation from the DNA-PK·DNA complex. Altogether, our data allow us to propose a model in which a DNA-PKcs-mediated phosphorylation is necessary both to activate Artemis endonuclease activity and to maintain its association with the DNA end site. This tight functional coupling between the activation of both DNA-PKcs and Artemis may avoid improper processing of DNA.

Effect of serum on in vitro electrically mediated gene delivery and expression in mammalian cells

In many cell systems, electric pulses can efficiently mediate gene transfer with a high level of expression in vitro. In vivo results have been reported where decrease in efficiency was obtained. The mechanisms involved in the process are unknown. Since, in vivo, the efficiency of non-viral methods of gene transfer is generally limited by the presence of serum, we report here the effect of serum on in vitro electrically mediated chinese hamster ovary cell membrane permeabilization, viability, gene transfer and expression. The results indicate that permeabilization and gene transfer are not inhibited by serum. By acting as a protector of cell viability, serum indeed increases gene transfer and expression.

Cellular localization of the molecular forms of acetylcholinesterase in primary cultures of rat sympathetic neurons and analysis of the secreted enzyme

Abstract: The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle‐conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60–80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane‐bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membranebound or entrapped in vesicles originating from, e.g., the Golgi apparatus.

IN VITRO AND EX VIVO ELECTRICALLY MEDIATED PERMEABILIZATION AND GENE TRANSFER IN MURINE MELANOMA

Abstract Electropulsation is a technique extensively used to introduce a large variety of compounds inside the cells or to obtain foreign gene expression. In that study, we compare in vitro and ex vivo permeabilization of murine melanoma by electric pulses. Penetration of a fluorescent dye, propidium iodide is used to detect permeabilization. Electric field strength inducing permeabilization is shown to depend on the shape of the cells, and varies from in vitro to ex vivo conditions. Penetration of a protein, β-galactosidase, and gene transfer can be obtained by pulsing the cells in the presence of the protein or of the plasmid DNA both in vitro and ex vivo. As for the permeabilization to small molecules, electric field conditions leading to macromolecules transfer depends on the characteristics of the cell culture.

In Vitro Delivery of Drugs and Other Molecules to Cells

The permeability of a cell membrane can be transiently increased locally when an external electric field pulse with an overcritical intensity is applied. A position dependent modulation of the membrane potential difference is induced during the pulse. A local membrane alteration is created, which may reseal. Its molecular definition remains unknown. This phenomenon is now commonly known as electroporation or electropermeabilization. The former term implies that physical pores are created in the lipid matrix. However their existence has never been clearly demonstrated. The term electroporation is therefore rather misleading.

Electrically Mediated Mammalian Cell Permeabilization to Macromolecules Application to Gene Transfer

Membrane permeabilization can be achieved by several methods such as microinjection (Diacumakos, 1978), use of polyethylene glycol or virus (Spear, 1987), particle bombardment (Klein et al., 1987). In the early 70’s, a physical method was proposed: electropermeabilization (Neumann and Rosenheck, 1972). An external electric field is applied to the cells. It induces the creation of a transmembrane potential which surrimposes to the natural potential difference of cells. When the resultant potential difference is brought higher than a threshold of 200–300mV, membrane becomes permeable (Teissie and Tsong, 1981; Teissie and Rols, 1993). Although the large use of electropulsation of cells in cellular biology and biotechnology (gene transfer (Neumann et al., 1982), cell hybridization, loading of cells with extra cellular molecules (Zimmermann et al., 1976)), and more recently in medecine (tissue electropermeabilization for gene therapy, cancer chemotherapy and transdermal drug delivery (Titomorov et al., 1991, Mir et al., 1991, Prautsnitz et al., 1993a)) molecular processes involved into the mechanism are still unknown.