Wu-Pong S, Weiss T L, Hunt C A
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Cell Mol Biol (Noisy-le-grand). 1994 Sep;40(6):843-50.
Because a major limitation of ODN (oligodeoxynucleotide) use is inefficient cellular uptake, methods to improve ODN uptake could have important implications in the investigational and possibly therapeutic use of ODNs. In this study, antisense c-myc ODN cellular uptake in elevated extracellular calcium was increased up to 48-fold in the four cell lines examined. The role of calcium in ODN cellular uptake was examined using a 21-base ODN complementary to the c-myc proto-oncogene and the Rauscher cells. Cells were pretreated with uptake inhibitors in either 1.8 (physiologic) or 5.4 mM calcium prior to addition of (32P) labelled ODN. In physiologic calcium conditions, ODN cellular uptake was partially dependent on cellular energy and a trypsin-sensitive surface protein. In contrast, in the presence of elevated (5.4 mM) extracellular calcium, trypsinization and metabolic inhibition had a reduced and no effect, respectively, on uptake. Endocytosis and lysosomotropic inhibitors did not decrease uptake in either calcium concentrations. Therefore, the mechanism of ODN uptake may depend on the level of extracellular calcium. Furthermore, surface binding accounted for approximately 60% of total uptake in both physiologic and elevated calcium concentrations, suggesting that the increased uptake was not due exclusively to increased surface binding. Thus, the predominant mechanism of ODN uptake may depend on the extracellular calcium concentration.
由于寡脱氧核苷酸（ODN）应用的一个主要限制是细胞摄取效率低下，因此改善ODN摄取的方法可能对ODN的研究及潜在治疗应用具有重要意义。在本研究中，在所检测的四种细胞系中，细胞外钙浓度升高时反义c-myc ODN的细胞摄取增加了48倍。使用与c-myc原癌基因互补的21碱基ODN和劳舍尔细胞研究了钙在ODN细胞摄取中的作用。在添加（32P）标记的ODN之前，先用1.8（生理浓度）或5.4 mM钙的摄取抑制剂预处理细胞。在生理钙条件下，ODN的细胞摄取部分依赖于细胞能量和一种对胰蛋白酶敏感的表面蛋白。相反，在细胞外钙浓度升高（5.4 mM）时，胰蛋白酶消化和代谢抑制对摄取的影响分别降低和无影响。内吞作用和溶酶体促效抑制剂在两种钙浓度下均未降低摄取。因此，ODN摄取机制可能取决于细胞外钙水平。此外，在生理和高钙浓度下，表面结合均占总摄取量的约60%，这表明摄取增加并非仅由于表面结合增加。因此，ODN摄取的主要机制可能取决于细胞外钙浓度。
