Characterization of the nuclear binding sites of oligodeoxyribonucleotides and their analogs.

The intracellular fate of antisense oligodeoxyribonucleotide (oligomers) is poorly understood. Recent observations strongly suggest that after endocytosis and escape from the endocytic compartments, oligomers accumulate in the nuclei of eukaryotic cells by simple diffusion. Isolated nuclei were used to determine the number of these nuclear binding sites and their affinity for phosphodiester, phosphorothioate, and methyl-phosphonate oligomers. These cell-free assays were correlated with intact cell microinjection experiments. Great differences have been observed between these analogs. These data are helpful in understanding the fate of oligomers and the mechanism of their action and could be helpful for a more rational design of antisense molecules.

Reversible inhibition of gene expression by a psoralen functionalized triple helix forming oligonucleotide in intact cells.

Triple helix formation of nucleic acids is the most rational approach to designing site-specific transcription inhibitors. To increase their efficiency, reactive moieties such as psoralen or ethenocytosine have been introduced on the third strand. In transfected cells, these compounds induce a site-specific covalent binding of the third strand to the targeted sequence and efficiently block RNA polymerases. However, the stability of this transcription inhibition has never been checked. We have designed a plasmid containing a triple helix binding site in the coding region of the beta-galactosidase reporter gene and a polymerase chain reaction assay to follow quantitatively the cross-link of a psoralen-derivatized third strand in transfected cells. This assay has revealed that the cross-link was removed within a few hours, leading only to a transitory inhibition of gene expression. Control experiments in DNA repair-deficient cells suggest the implication of repair enzymes in this process.

Nuclear location of synthetic oligonucleotides microinjected somatic cells: its implication in an antisense strategy.

Synthetic oligonucleotides are widely used to modulate gene expression. However their development as genetic tools and as molecule of therapeutic interest is restricted by the poor knowledge of their mechanism of action and of their uptake by cells. We recently found that microinjected oligonucleotides accumulates in the nucleus of the cells. These observations are described and their implications are discussed.

[Control of gene expression by antisense nucleic acids]. / Contrôle de l'expression génétique par des acides nucléiques anti-sens.

The use of antisense RNA or of antisense oligonucleotides for the specific control of viral or cellular genes expression has undergone rapid developments recently; their respective advantages and drawbacks will be discussed. Progresses in oligonucleotides chemistry have lead to the synthesis of analogs with improved pharmacological properties. Besides the antisense approach, which usually targets translation initiation or splicing sites, it is possible to interfere specifically with gene expression through triple helix formation (anti-gene strategy) or through the titration of regulatory proteins (sense approach). A major problem encountered in the use of synthetic oligonucleotides is their delivery to their nuclear or cytoplasmic targets after cell uptake by an endocytic pathway; our own work in this field will be discussed. Finally, we will describe the strategies followed by our group to improve the bioavailability of antisense oligonucleotides, as for instance conjugation to poly (L-lysine) or encapsidation in antibody-targeted liposomes.

N-myristoyl-transferase activity in cancer cells. Solubilization, specificity and enzymatic inhibition of a N-myristoyl transferase from L1210 microsomes.

The activity catalyzed by N-myristoyl transferase (NMT) is described for the first time in microsome-rich fractions from the murine leukemia cell line L1210, rat brain and mouse liver as biological sources. The enzyme from each source can accommodate various types of proteins (protein kinase A, virus structural gag protein or pp60src) as modelized by the use of their N-terminal derived peptides (GNAAAARR, GQTVTTPL and GSSKSKPKDP, respectively). As for some other types of membrane-bound enzymes, NMT activity can be enhanced by pretreatment with various types of detergents, amongst which Triton 770 and deoxycholate were the most potent. Further experiments on the L1210 microsome-rich fractions demonstrate that these two detergents were able to solubilize the microsomal enzyme, without modifying its substrate specificity. Finally, three compounds described in the literature to be inhibitors of NMT activity from other sources were tested for L1210 microsome-associated activity. None of them show any significant potency in inhibiting this activity. A new compound, myristoylphenylalanine, shows a slightly better inhibitory effect on the L1210 microsomal activity than the reference compounds with a median inhibitory concentration (IC50) of 0.2 mM.