Parameters that affect cellular transfection as accomplished by introducing DNA via carriers composed of cationic synthetic amphiphiles, have been investigated, with the aim to obtain insight into the mechanism of DNA translocation. Such insight may be exploited in optimizing carrier properties of synthetic amphiphiles for molecules other than nucleic acids. In the present work, the interaction of vesicles composed of the cationic amphiphile dioleyloxy-propyl-trimethylammonium chloride (DOTMA) with cultured cells was examined. The results show that optimal transfection is dependent on the concentration of lipid, which determines the efficiency of vesicle interaction with the target cell membrane, as well as the toxicity of the amphiphiles towards the cell. A low lipid/DNA ratio prevents the complex from interacting with the cell surface, whereas at a relatively high amphiphile concentration the complex becomes toxic. Translocation efficiency is independent of the initial vesicle size but is affected by the size of the DNA. An incubation time of the DNA/amphiphile complex and cells of approx. 2-4 h is required for obtaining efficient transfection. In conjunction with observations on DNA/amphiphile complex-induced hemolysis of erythrocytes, a mechanism of DNA-entry is proposed which involves translocation of the nucleic acids through pores across the membranes rather than delivery via fusion or endocytosis. Dioleoylphosphatidylethanolamine, a phospholipid frequently used in a mixture with DOTMA ('lipofectin') strongly facilitates this pore formation. Translocation of the DNA is effectively prevented when the cells are pretreated with Ca2+ or pronase. These observations suggest that Ca(2+)-sensitive cell surface proteins play a role in amphiphile-mediated DNA translocation.
The possibility that transport of proteolipid protein (PLP) from its site of synthesis to the plasma membrane is dependent on cotransport with (sulfo)galacto-cerebrosides was investigated in primary cultured oligodendrocytes and Chinese hamster ovary (CHO) cells expressing PLP. Sulfation was inhibited by growing oligodendrocytes in the presence of a competitive inhibitor of this process, sodium chlorate. Under these circumstances, sulfatide synthesis was inhibited by 85%. Nevertheless, PLP was still delivered to the plasma membrane in quantitative amounts. Furthermore, when PLP was expressed in CHO cells, which normally synthesize very low amounts of galactosyl ceramide (GalCer) and no sulfatide, PLP was transported to the plasma membrane. Moreover, in CHO cells coexpressing PLP and ceramide galactosyl transferase, PLP cell surface labeling was unaltered. Noting that it has been demonstrated that proteins destined for the apical surface of epithelial cells colocalize with glycolipid-enriched microdomains, we isolated detergent-insoluble membrane complexes from cultured oligodendrocytes. We found, however, that most of the PLP is present in the detergent-soluble fraction and, furthermore, that PLP could not be chased into or out of the insoluble fraction. Taken together, these data make it very likely that in oligodendrocytes PLP transport takes place irrespective of the presence of glycosphingolipids GalCer and sulfatide.
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