par Ouali, Mustapha 
Président du jury Droogmans, Louis
Promoteur Ruysschaert, Jean Marie
Publication Non publié, 2007-02-15
Président du jury Droogmans, Louis
Promoteur Ruysschaert, Jean Marie
Publication Non publié, 2007-02-15
Thèse de doctorat
| Résumé : | Cationic lipids are efficient tools to introduce nucleic acids and proteins into cells. Elucidation of the mechanism and cellular pathways associated to such a transport has been relatively slow, even though significant progress has been made in the characterization of the intracellular trafficking of cationic lipid/DNA complexes. Surprisingly, little is known about the effects of these delivery vectors on cell functioning. In the present thesis, we show that cationic lipids and cationic lipid/DNA complexes strongly increase the intracellular Ca2+ concentration. The end point of the Ca2+ increase was ~400 nM from a basal level of ~100 nM. The [Ca2+]i increase was studied using K562 and Jurkat cells cultured in vitro. This effect is weakened following addition of DNA to cationic liposomes, although remaining very large at cationic lipid/DNA ratios commonly used for cell transfection experiments. Removal of extracellular Ca2+ did not abolish this effect significantly and preincubating K562 cells with the Ca2+-ATPase inhibitor thapsigargin strongly abolished intracellular Ca2+ concentration increase, indicating that Ca2+ was released mainly from internal Ca2+ stores sensitive to thapsigargin. Pretreatment of the cells with the phospholipase C inhibitor U73122 blocked the intracellular Ca2+ concentration rise, suggesting an inositol pathway-dependent mechanism. LDH release assay indicates that in the conditions used for fluorescence measurement and in those used to transfer DNA into cells, cationic liposomes diC14-amidine and DOTAP had no massive cytotoxic effects. Cationic liposomes showed more toxicity than their corresponding complexes; this toxicity decreases in the presence of serum. The effect of cationic lipids on phosphatidylinositol-specific phospholipase C (PI-PLC) was quantitatively assessed using phosphatidylinositol (PI) and radiolabeled phosphatidylinositol ([3H]-PI). Incorporation of diC14-amidine into PC/PI vesicle activated PI-PLC and was shown to activate the hydrolysis of PI and [3H]-PI. Our data may suggest that mobilization of intracellular Ca2+ by complex could have an effect on the transfection process itself. These results indicate for the first time that cationic lipids and cationic lipid/DNA complexes are not inert and can affect the functioning of the cells by increasing their intracellular Ca2+. |
