Calcium dependent cellular uptake of a c-myc antisense oligonucleotide.

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.

Antisense c-myc oligodeoxyribonucleotide cellular uptake.

Antisense oligonucleotides have therapeutic potential as inhibitors of gene expression. However, the mechanism by which an intact oligonucleotide reaches the intracellular site of action is unknown. In this study, we use an oligodeoxyribonucleotide 21-mer complementary to the translation initiation codon of the c-myc protooncogene to study the mechanism of oligonucleotide uptake and internalization into Rauscher Red 5-1.5 cells. We find trypsin-sensitive and trypsin-insensitive surface binding, in addition to internalization. Uptake is partially energy dependent and inhibited by charged molecules, including DNA, ATP, a random sequence oligonucleotide, and dextran sulfate. Uptake does not appear to occur via a traditional receptor-mediated uptake pathway because chloroquine, monensin, and phenylarsine oxide pretreatment does not significantly decrease internalization. An anion channel inhibitor, SITS, and the salts, NaCl, Na2SO4, and NH4Cl, significantly decrease oligonucleotide uptake. Whether uptake occurs via a channel or a novel uptake mechanism is still unknown. A model is proposed which reasonably simulates the experimental data.

Cholecystokinin innervation of rat thalamus, including fibers to ventroposterolateral nucleus from dorsal column nuclei.

The distribution of cholecystokinin octapeptide immunoreactive fibers and puncta in the adult rat thalamus was studied using immunocytochemical methods. Small to moderate numbers of immunoreactive fibers were present in the lateral habenular nucleus, ventral lateral geniculate nucleus, zona incerta, parataenial, mediodorsal, medioventral, and submedial nuclei, the rhomboid, paracentral, central lateral and parafascicular nuclei, and in the medial geniculate and dorsal lateral geniculate nuclei. Moderate to large numbers of cholecystokinin (CCK)-positive fibers were present in the paraventricular nuclei, the reticular nucleus, the anteroventral, anteromedial, and central medial nuclei, and in the rostral extension of the internal medullary lamina between the parataenial and anteroventral nuclei. Dense concentrations of immunoreactive fibers were also found in a principal sensory relay nucleus, the ventroposterolateral nucleus (VPL), of the ventrobasal complex. The number of CCK-positive fibers in VPL showed a marked unilateral decrease in rats which had received lesions of the contralateral gracile and cuneate nuclei. The results of this study demonstrate that CCK-immunoreactive fibers and puncta are widely distributed in the rat thalamus, and that the source of these fibers in VPL is probably the dorsal column nuclei.