CALCIUM-DEPENDENT OLIGONUCLEOTIDE CELLULAR UPTAKE

Oligodeoxynucleotides (ONs) are currently being tested in clinical tri als as anti-viral and thrombolytic agents. Although ONs are biological ly active in almost every cell line examined, these molecules are inef ficiently 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 necessar y for the rational design of optimization of ON cellular uptake, deliv ery, 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 m ouse 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 mech anism 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 determ ine whether changes in intracellular free calcium [Ca](i) may influenc e ON uptake, [Ca](i) was either increased or decreased prior to additi on of the ON. Second, to determine whether protein kinase C or calmodu lin are involved in uptake, the activity of each of these proteins was either increased or decreased. Neither altering [Ca](i) or the activi ty of protein kinase C or calmodulin altered the extent of ON uptake i n these cells. Finally, the effect of other cations were tested for th eir ability to increase ON uptake. Lanthanum and cadmium increased upt ake whereas sodium and magnesium had no effect. These findings suggest that cation-mediated ON cellular uptake may not result from alteratio n of a calcium-dependent biological process.