Belting M, Petersson P
Department of Cell and Molecular Biology, Section for Cell and Matrix Biology, Lund University, P.O.B. 94, S-221 00 Lund, Sweden.
Biochem J. 1999 Sep 1;342 ( Pt 2)(Pt 2):281-6.
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.
先前的研究已经表明，阳离子脂质介导的基因转移对蛋白聚糖存在依赖性[米斯利克和巴尔德施维勒（1996年）《美国国家科学院院刊》93卷，第12349 - 12354页；蒙克斯、钟、西普雷斯 - 帕拉辛、希思和德布斯（1998年）《生物化学杂志》273卷，第26164 - 26170页]。我们已经评估了蛋白聚糖参与阳离子脂质介导的基因转移的机制。在野生型中国仓鼠卵巢（CHO）细胞（CHO - K1）、硫酸乙酰肝素缺陷型CHO细胞（pgsD - 677）和蛋白聚糖缺陷型CHO细胞（pgsB - 618）中研究了DNA质粒摄取和基因表达。在阳离子脂质与DNA的最佳比例下，与硫酸乙酰肝素缺陷型细胞和野生型细胞相比，在蛋白聚糖缺陷型细胞中观察到报告基因表达大幅下降。然而，当通过电穿孔转染时，各细胞系之间的报告基因表达没有差异。此外，通过测量细胞内（32）P标记和罗丹明标记的DNA质粒评估，所有细胞系均表现出相等的阳离子脂质 - DNA复合物摄取活性。对野生型和蛋白聚糖缺陷型细胞的反射光图像分析表明，阳离子脂质对蛋白聚糖缺陷型细胞具有优先毒性。细胞生长测定证实了这一点，表明阳离子脂质在蛋白聚糖缺陷型细胞和氯酸盐处理的野生型细胞中比在其他细胞系中表现出更大的抗增殖活性。添加外源性硫酸化糖胺聚糖可消除阳离子脂质的生长抑制作用。我们得出结论，蛋白聚糖的糖胺聚糖部分对阳离子脂质细胞毒性起到保护作用，从而在体外实现最佳转染效率。
