Henderson G B, Stein C A
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037, USA.
Nucleic Acids Res. 1995 Sep 25;23(18):3726-31. doi: 10.1093/nar/23.18.3726.
Polyanionic 5'-cholesteryl-phosphorothioate oligodeoxynucleotides of varying polymer length and nucleobase composition were examined for an effect on methotrexate transport via the reduced-folate carrier of L1210 mouse cells. Methotrexate transport was inhibited by each of the oligodeoxynucleotide analogs tested. Inhibition was most pronounced (IC50 = 0.21 microM, standard assay) for a 5'-cholesteryl heteropolymer consisting of 15 phosphorothioate deoxynucleotides with alternating deoxycytosine and deoxyadenosine (Chol-PS-d(CA)7C). Homopolymers with 15 deoxycytosine (Chol-PS-dC15) or deoxythymidine (Chol-PS-dT15) residues were approximately 2-fold less inhibitory than Chol-PS-d(CA)7C. The relative potency of transport inhibition by deoxycytosine oligomers of varying length was: Chol-PS-dC5 > Chol-PS-dC15 > Chol-PS-dC28 > Chol-PS-dC3. Substantial inhibition was retained in cells preincubated with inhibitors and washed prior to transport determinations and the inhibitor sensitivity could be increased substantially by reducing the concentration of cells. Mixed competitive and non-competitive inhibition was observed for each analog. In standard high-folate medium, Chol-PS-oligodeoxynucleotides (5.0 microM) had minimal effects on the growth of L1210 cells, but antagonized the cytotoxicity of methotrexate. The response to methotrexate (IC50 = 12 nM) decreased to the greatest extent (20.8-fold) in the presence of Chol-PS-d(CA)7C (IC50 = 250 nM). Under limiting folate conditions, Chol-PS-d(CA)7C alone inhibited cells growth by a process which could be reversed by folic acid. The results show that Chol-PS-oligodeoxynucleotides are among the most potent known inhibitors of the reduced-folate carrier. Direct growth inhibition of folate-deficient cells and antagonism of methotrexate cytotoxicity indicate that Chol-PS-oligodeoxynucleotides retain the ability to inhibit the reduced-folate carrier for several days in cultured cells.
研究了不同聚合物长度和核碱基组成的聚阴离子5'-胆固醇基硫代磷酸酯寡脱氧核苷酸对甲氨蝶呤通过L1210小鼠细胞的还原型叶酸载体转运的影响。所测试的每种寡脱氧核苷酸类似物均抑制甲氨蝶呤的转运。对于由15个硫代磷酸酯脱氧核苷酸组成的5'-胆固醇基杂聚物，其中脱氧胞嘧啶和脱氧腺苷交替排列（Chol-PS-d(CA)7C），抑制作用最为明显（IC50 = 0.21 microM，标准测定法）。具有15个脱氧胞嘧啶（Chol-PS-dC15）或脱氧胸苷（Chol-PS-dT15）残基的同聚物的抑制作用比Chol-PS-d(CA)7C约低2倍。不同长度的脱氧胞嘧啶寡聚物对转运抑制的相对效力为：Chol-PS-dC5 > Chol-PS-dC15 > Chol-PS-dC28 > Chol-PS-dC3。在用抑制剂预孵育并在转运测定前洗涤的细胞中仍保留有显著的抑制作用，并且通过降低细胞浓度可大幅提高抑制剂敏感性。对每种类似物均观察到混合竞争性和非竞争性抑制作用。在标准高叶酸培养基中，Chol-PS-寡脱氧核苷酸（5.0 microM）对L1210细胞的生长影响极小，但可拮抗甲氨蝶呤的细胞毒性。在Chol-PS-d(CA)7C（IC50 = 250 nM）存在下，对甲氨蝶呤的反应（IC50 = 12 nM）下降幅度最大（20.8倍）。在叶酸限制条件下，单独的Chol-PS-d(CA)7C通过一种可被叶酸逆转的过程抑制细胞生长。结果表明，Chol-PS-寡脱氧核苷酸是已知的对还原型叶酸载体最有效的抑制剂之一。对叶酸缺乏细胞的直接生长抑制作用以及对甲氨蝶呤细胞毒性的拮抗作用表明，Chol-PS-寡脱氧核苷酸在培养细胞中保留了抑制还原型叶酸载体数天的能力。
