Protective role for proteoglycans against cationic lipid cytotoxicity allowing optimal transfection efficiency in vitro.
A dependence on proteoglycans for cationic lipid-mediated gene transfer has been suggested in previous studies [Mislick and Baldeschwieler (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12349-12354; Mounkes, Zhong, Cipres-Palacin, Heath and Debs (1998) J. Biol. Chem. 273, 26164-26170]. We have evaluated the mechanism of proteoglycan involvement in cationic lipid-mediated gene transfer. DNA plasmid uptake and gene expression were studied in wild-type Chinese hamster ovary (CHO) cells (CHO-K1), heparan sulphate-deficient CHO cells (pgsD-677) and proteoglycan-deficient CHO cells (pgsB-618). At an optimal ratio of cationic lipid to DNA, a substantial decrease in reporter gene expression was observed in proteoglycan-deficient cells compared with that in heparan sulphate-deficient and wild-type cells. However, there were no differences in reporter gene expression between the cell lines when transfected by electroporation. Moreover, all cell lines exhibited equal cationic-lipid-DNA complex uptake activities, as assessed by the measurement of intracellular (32)P-labelled and rhodamine-labelled DNA plasmid. An analysis of reflected-light images of wild-type and proteoglycan-deficient cells suggested that cationic lipids were preferentially toxic to proteoglycan-deficient cells. Cell-growth assays confirmed this, showing that cationic lipids exhibited a greater anti-proliferative activity in proteoglycan-deficient cells and in chlorate-treated wild-type cells than in the other cell lines. The growth-inhibitory effect of cationic lipids was abrogated by the addition of exogenous sulphated glycosaminoglycans. We conclude that the glycosaminoglycan part of proteoglycans serves a protective role against cationic lipid cytotoxicity, allowing optimal transfection efficiency in vitro.
Belting
M
M
Department of Cell and Molecular Biology, Section for Cell and Matrix Biology, Lund University, P.O.B. 94, S-221 00 Lund, Sweden. mattias.belting@medkem.lu.se
Petersson
P
P
D000818:Animals; D016466:CHO Cells; D002412:Cations; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D018274:Electroporation; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D006025:Glycosaminoglycans / Q000494:pharmacology; D008055:Lipids / Q000737:chemistry / Q000633:toxicity*; D008081:Liposomes; D008156:Luciferases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011509:Proteoglycans / Q000172:deficiency / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection / Q000187:drug effects* / Q000379:methods*
D002412:Cations; D006025:Glycosaminoglycans; D008055:Lipids; D008081:Liposomes; D011509:Proteoglycans; D008156:Luciferases