Gao W Y, Storm C, Egan W, Cheng Y C
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510.
Mol Pharmacol. 1993 Jan;43(1):45-50.
The uptake and distribution of phosphorothioate oligodeoxynucleotides by human cells were studied using 35S-labeled 28-mer phosphorothioate oligodeoxycytidine [S-(dC)28]. Accumulation of intracellular S-(dC)28 was found to be higher in the carcinoma cells (grown in monolayers) than in the leukemia cells (grown in suspension culture). A hepatoma cell line transfected with hepatitis B virus, 2215, was chosen for further studies. The uptake of S-(dC)28 was partially dependent on temperature and energy. The intracellular concentration was significantly higher than that in the medium and the amount accumulated was dependent on the extracellular concentration. It appears that the uptake of S-(dC)28 involves mechanisms of both fluid-phase pinocytosis and adsorptive endocytosis. Neither oligonucleotides nor 5'-phosphorylated nucleotides inhibited S-(dC)28 uptake. Unlike horseradish peroxidase, which was primarily associated with endosomes once it was taken into the cell, S-(dC)28 was found to be present in both nuclear and cytoplasmic fractions. Efflux of S-(dC)28 from the cell was multiphasic; a trapping mechanism that could be due to a potent interaction of S-(dC)28 with cellular proteins was implicated. This trapping mechanism could be responsible for the lack of biological activity such as cytotoxicity and antisense activity of phosphorothioate oligodeoxynucleotides in some human cells.
利用35S标记的28聚硫代磷酸寡脱氧胞苷[S-(dC)28]研究了人细胞对硫代磷酸寡脱氧核苷酸的摄取和分布。发现癌细胞（单层生长）中细胞内S-(dC)28的积累高于白血病细胞（悬浮培养）。选择转染了乙肝病毒的肝癌细胞系2215进行进一步研究。S-(dC)28的摄取部分依赖于温度和能量。细胞内浓度显著高于培养基中的浓度，积累量取决于细胞外浓度。似乎S-(dC)28的摄取涉及液相胞饮作用和吸附性内吞作用两种机制。寡核苷酸和5'-磷酸化核苷酸均不抑制S-(dC)28的摄取。与辣根过氧化物酶不同，辣根过氧化物酶一旦进入细胞主要与内体相关，而S-(dC)28在细胞核和细胞质组分中均有发现。S-(dC)28从细胞中的流出是多相的；涉及一种捕获机制，这可能是由于S-(dC)28与细胞蛋白的强相互作用所致。这种捕获机制可能是硫代磷酸寡脱氧核苷酸在某些人细胞中缺乏细胞毒性和反义活性等生物学活性的原因。
