Calcium-dependent oligonucleotide cellular uptake.
Oligodeoxynucleotides (ONs) are currently being tested in clinical trials as anti-viral and thrombolytic agents. Although ONs are biologically active in almost every cell line examined, these molecules are inefficiently internalized. In addition, the mechanism of ON transport to the active site in either the cytoplasm or nucleus remains ill-defined. Thus, elucidation of the mechanism of ON cellular uptake is necessary for the rational design of optimization of ON cellular uptake, delivery, and targeting. Previous studies in this laboratory have examined the mechanism of ON cellular uptake using a phosphodiester ON which is both biologically active and internalized by the Rauscher Red 5-1.5 mouse erythroleukemia cell line. The uptake is calcium-dependent since uptake is significantly reduced in calcium-free media and enhanced up to 20-fold when incubated in elevated calcium concentrations by a mechanism which may differ from uptake in physiologic calcium. Therefore, the goal of this paper is to further examine the mechanism of calcium-dependent phosphodiester ON uptake in Rauscher cells. First, to determine whether changes in intracellular free calcium [Ca]i may influence ON uptake, [Ca]i was either increased or decreased prior to addition of the ON. Second, to determine whether protein kinase C or calmodulin are involved in uptake, the activity of each of these proteins was either increased or decreased. Neither altering [Ca]i or the activity of protein kinase C or calmodulin altered the extent of ON uptake in these cells. Finally, the effect of other cations were tested for their ability to increase ON uptake. Lanthanum and cadmium increased uptake whereas sodium and magnesium had no effect. These findings suggest that cation-mediated ON cellular uptake may not result from alteration of a calcium-dependent biological process.
Wu-Pong
S
S
Department of Pharmacy and Pharmaceutics, Virginia Commonwealth University, Richmond 23298, USA.
D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D002118:Calcium