Eric Hebert

Improvement of exogenous DNA nuclear importation by nuclear localization signal-bearing vectors: a promising way for non-viral gene therapy?

Abstract Several vectors have been developed in order to target genes to specific cells. Virus‐based vectors lead to a high transfection efficiency in vitro, but display important disadvantages such as pathological risks, which they expose to patients. Plasmid‐associated chemical vectors lack these disadvantages, but allow only a very low efficiency of transgene expression. Most of the non‐viral‐based gene transfer techniques developed until now mainly focused their efforts to overcome the problem of DNA entry into the cell. Some recent works, however, have begun to investigate the nucleus entry problem and suggest that the trafficking of DNA from cytosol to the nucleus may be improved by using the nuclear localization signal (NLS) found in some nuclear proteins. If the vector contains one or several NLS, either as covalently or non‐covalently DNA‐linked peptides, a competition may take place between the rate of dissociation of the DNA‐vector complexes and the rate of loading of the complexes to the NLS‐mediated nucleus importation machinery. This equilibrium may be displaced towards the importation pathway by the use of NLS‐bearing proteins instead of peptides. The possibility of recruiting normal endogenous cellular pathways of nuclear uptake to promote entry of exogenously applied DNA through the nuclear pore complex would, thus, seem promising. Nevertheless, attempts to improve the transport of DNA to the nucleus through the use of NLSs have achieved limited success. Although these systems show improved transgene expression, little is known about how they function in transfected cells, and the optimal formulation for gene expression is yet to be determined.

Endogenous Lectins as Cell Surface Transducers

Interactions between cells or between cell and substratum involve specificreceptors and their ligands. Among the various cell surface receptorsidentified during the last decades, the carbohydrate-binding proteins,e.g., lectins are of peculiar interest because glycolipids, glycoproteinsand proteoglycans have been shown to interact with lectins on the surfaceof animal cells. Animal lectins are recognized as molecules playingimportant roles in a variety of biological processes through binding toglycoconjugates and lectin-like receptors such as selectins, sialoadhesins(CD22, CD33), natural killer receptors (NKR-P1, CD69 and CD94/NKG2),hyaluronate receptors (CD44, RHAMM, ICAM-1), B-cell associated antigen(CD23, CD72), β2 leukocyte integrin (CD11b/CD18) or the well-knownreceptors for mannose, mannose-6-phosphate or asialoglycoprotein havebeen suggested to be able to mediate the transfer of information fromthe outside to the inside of the cell. This review focuses on the mostrecent advances in our understanding of the molecular basis of“outside-in” signaling mediated by lectins. Lectin-likereceptors are involved in signal transduction in a great variety of ways;at the molecular level, they mimic in most of the cases the function ofgrowth factor receptor either coupled to tyrosine kinase activity or toheterotrimeric G protein. They lead to a multiplicity of cellular eventsfollowing their activation depending on factors such as cellular type,species and/or tissue. Nevertheless the potential of surface lectins astransducers is emphasized by the observation that in a few cases lectin-likereceptors induce either novel signal transduction mechanism or newintracellular events with regards to what it has been observed as aconsequence of growth factor receptor activation. This observation bringsthe idea that lectins may offer, as cell surface transducers, an alternativeor additional signaling potential to cell.

Hyperreactivity of the B-Z Junctions Probed by Two Aromatic Chemical Carcinogens, 2-N,N-Acetoxyacetylaminofluorene and 3-N,N-Acetoxyacetyl-Amino-4,6-Dimethyldipyrido[1,2-a:3′,2′-d] Imidazole

The reaction between DNAs in various conformations and two isosteric chemical carcinogens 2-N,N–cetoxyacetylaminofluorene and 3-N,N–cetoxya-cetylamino-4,6-dimethyldipyrido[1,2–:3′,2′-d] imidazole has been studied. The modification of DNA has been analysed at the nucleotide level by means of the 3′–5′ exonuclease activity of T4 DNA polymerase. Both carcinogens bind covalently to (dC-dG)16 and (dG-dT)15 sequences inserted in closed circular plasmids when the inserts are in the B form; they do not bind to these inserts when they are in the Z form. The reactivity of guanine residues at the B-Z junction depends upon the superhelical density of the plasmids and upon the base sequence at the junction. A strong hyperreactivity is observed on the 3′ side of the (dC-dG)16 in pLP32, the insert being in the Z form. It is concluded that the reactivity of guanine residues with both carcinogens depends upon the DNA conformation. The non-reactivity of Z-DNA and the hyperreactivity of some sequences under topological stress might have some importance in chemical carcinogenesis.

Model Studies on the in Vitro Activation of c-Ha-ras Protooncogene by Dietary Components

Evidence for the activation of ras protooncogenes by chemical carcinogens is provided both by the reproducible activation observed in chemically-induced animal tumors (reviewed in 1) and by our demonstration that in vitro modification of ras protooncogenes with ultimate carcinogens results in transformed foci on transfection into NIH3T3 cells2,3. In the present study, transforming oncogenes have been generated by the in vitro modification of plasmid containing human c-Ha-ras with the model ultimate carcinogens 3-N, N-acetoxyacetylamino-4,6-dimethyl-dipyrido[1,2-a:3′,2′-d]imidazole (N-AcO-AGlu-P-3) and 1′-acetoxysafrole (AcO-safrole). The mutations produced have been analyzed by selective amplification of the regions surrounding codons 12 and 61 and subsequent oligonucleotide hybridization.