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S Wu-Pong

Publications and source records attributed to S Wu-Pong.

15 recordsLinked to original sources
PubMed2000

Alternative interpretations of the oligonucleotide transport literature: insights from nature.

Elucidation of the mechanism of oligonucleotide (ON) cellular internalization has met an impasse at the lipid penetration stage. ON internalization is commonly regarded to involve endocytosis, yet the method by which the ON penetrates the endosome membrane remains a mystery despite more than 10 years of research by multiple laboratories. In addition, the literature regarding this topic is fraught with discrepancies and inconsistencies. Therefore, the goal of this review is to propose and illustrate the feasibility of the notion that the literature discrepancies are perhaps an indication of a complex transport mechanism involving more than one uptake pathway. Accordingly, ON- and cell-differences in uptake may be attributed to differences in the relative importance of these pathways for different cell types and ONs. An example of one such pathway is reviewed and critiqued in this communication with respect to its hypothetical role in ON uptake. Other innovative mechanisms should similarly be considered to stimulate new ideas, discussion and research in this unique and interesting field.

Animals↗
PubMed1999

Oligonucleotide transport in rat and human intestine ussing chamber models.

Cellular and intestinal absorption of naked oligonucleotides (ONs) is limited and still remains a developmental challenge. A previous report in the literature suggests that ON absorption occurs via a paracellular mechanism. The aim of this study was to test this hypothesis using rat and human intestine in a Ussing chamber and in Caco-2 cells. Transport of a (35)S-labelled mixed backbone ON (MBO) across human or rat intestinal tissue or across Caco-2 cells was measured after a 2-h incubation in the presence or absence of increasing MBO concentrations or with uptake inhibitors and enhancers. MBO intestinal absorption was compared with an internal standard, mannitol. (35)S-MBO demonstrated very little absorption (<1%) across rat and human intestinal tissues. Transport appeared to be unsaturable up to 500 microM, and relatively insensitive to compounds that opened tight junctions or inhibited P-glycoprotein. However, preliminary studies with Caco-2 cells suggest a possible saturable mechanism at higher ON concentrations. Confocal fluorescence microscopy studies show that fluorescein isothiocyanate (FITC)-MBO was internalized into intestinal cells. Although some differences in ON transport were observed as a function of the transport model, MBO transport was mostly consistent with a transcellular, rather than a paracellular, absorption mechanism.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Increased potency of an aptameric G-rich oligonucleotide is associated with novel functional properties of phosphorothioate linkages.

We previously showed that inhibition of the expression of CD28 (an essential immune receptor on T cells) mediated by a phosphorothioate (PS)-modified aptameric oligodeoxynucleotide (ODN) sequence, GR1, resulted in reduced T cell responses in vitro and in vivo. Using GR1 sequences differing only in the amount of terminal PS linkages (chimeric SO-ODN), the present study demonstrated that even after a substantial reduction in PS linkages, this 18-mer ODN sequence could still confer functionality in the ODN-mediated inhibition of CD28 expression. We showed that secondary structure and full retention of the ability to form a specific protein-ODN complex and to increase cellular uptake in activated Jurkat T cells were critical parameters in the determination of the magnitude of bioactivity of chimeric SO-ODN. We report that a chimeric SO-ODN with terminal PS linkages that total 9 (ICN 17221) or 12 (ICN 17263) was sufficient to inhibit CD28 expression and suppress in vivo inflammatory ear responses to contact allergen in mice with similar potency to the 17-thioate S-ODN (ICN 16064). Interestingly, all chimeric SO-ODN showed similar in vitro nuclease resistance. These data suggest alternate functional properties for PS linkages, unrelated to nuclease resistance, in enhancing the bioactivity of a G-rich aptamer.

Animals↗
PubMed1999

Alterations in methadone metabolism during late pregnancy.

Traditionally, methadone maintenance therapy has been a once-daily dosing schedule. The current study evaluates the effectiveness of this regimen during pregnancy. A total of 23 pregnant and 16 non-pregnant opioid-dependent patients were studied in two phases to evaluate pregnancy-dependent changes in methadone pharmacokinetics. In the first phase, pregnant patients had a statistically significant higher elimination rate constant (k) and lower half-life compared to non-pregnant controls. In the second phase, the apparent clearance (Cl/F) was significantly greater during pregnancy, with preliminary data suggesting that this observation results from a decrease in the fraction of dose absorbed (F). The implications of these findings on dosing regimens during pregnancy is discussed.

Adult↗
PubMed1997

Potentiation of ara-C-induced apoptosis by the protein kinase C activator bryostatin 1 in human leukemia cells (HL-60) involves a process dependent upon c-Myc.

The role of the nuclear phosphoprotein c-Myc has been examined with respect to the regulation of 1-beta-D-arabinofuranosylcytosine (ara-C)-induced apoptosis in human leukemia cells exposed to bryostatin 1 and other pharmacologic protein kinase C (PKC) activators. Pretreatment of HL-60 cells for 24 hr with 10 nM bryostatin 1 significantly potentiated the ability of ara-C (10 microM; 6 hr) to induce apoptosis without reducing the expression of c-Myc protein. In contrast, equivalent exposure to the stage 2 tumor-promoting PKC activator mezerein (10 nM) in conjunction with ara-C reduced c-Myc levels by 87% and failed to potentiate apoptosis. Co-administration of bryostatin 1 with mezerein before ara-C prevented down-regulation of c-Myc and augmented cell death, whereas co-treatment with the calcium ionophore A23187 (250 nM) and bryostatin 1 reduced c-Myc levels by 80% and abrogated the increase in ara-C-induced apoptosis. When cells were exposed for 24 hr to a c-myc antisense oligonucleotide (AS-ODN;10 microM) but not to a scrambled sequence ODN (SS-ODN) prior to ara-C, c-Myc expression was reduced by 81%, and apoptosis and cell viability were unperturbed. However, AS-ODN (but not SS-ODN) reduced c-Myc protein in cells pre-exposed to bryostatin 1 by 74% and abrogated potentiation of ara-C-induced apoptosis. The actions of c-myc AS-ODN did not stem from proximal G1 arrest/differentiation or biochemical events, since they were not associated with a reduction in the S-phase cell fraction, p21(WAF1/CIP1) induction, pRb hypophosphorylation, or alterations in ara-C metabolism. Together, these findings indicate that HL-60 cell apoptosis proceeds by both c-Myc-dependent and -independent pathways, and that only the former are involved in the potentiation of ara-C-mediated cell death by bryostatin 1.

Antineoplastic Agents↗
PubMed1997

Oligonucleotide biological activity: relationship to the cell cycle and nuclear transport.

Previous studies suggest that oligodeoxynucleotide (ODN) cellular uptake is cell cycle-dependent which may have important implications in cancer cell targeting. To further our understanding of ODN transport and activity, this study examines the relationships between the cell cycle, ODN cellular uptake, intracellular transport, and activity. An antisense c-myc ODN 21-mer was used to study ODN cellular uptake in Rauscher erythroleukemia cells synchronized by either chemical methods or flow cytometry. ODN uptake was examined using subcellular fractionation and confocal fluorescence microscopy. Western blot analysis was used to measure ODN-mediated decreases in c-myc protein levels. Intracellular ODN distribution and extent of uptake was influenced by the phase of the cell cycle, but the mechanism of uptake was not. The relative activity of the antisense ODN was positively correlated to ODN distribution to the cytosol, but negatively correlated to total cellular uptake. Although ODN total cellular uptake is positively influenced by the cell cycle, retention of the ODN in the cytosol (presumably extra-vesicularly) appeared to be relevant in determining the activity of an antisense ODN. Novel methods to target cytosol-acting drugs to the cytoplasm may therefore be warranted.

Biological Transport↗
PubMed1996

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.

Base Sequence↗
PubMed1996

The role of multivalent cations in oligonucleotide cellular uptake.

Oligodeoxynucleotides (ONs) are a powerful new class of drugs whose transport to the cytoplasm or nucleus inefficient and incompletely defined. Thus, to further extend our understanding of the mechanism of ON cellular uptake, the role of multivalent cations in ON cellular uptake was examined. All the multivalent cations tested, except magnesium, significantly increase ON uptake and was predictably related to the cation's electronegativity but only partially due to an increase in surface-binding. In addition, the primary mechanism of uptake is largely independent of trypsin-sensitive surface components, cellular energy, and several specific, calcium-dependent cellular processes. Therefore, ON cellular uptake may involve an interaction between multivalent cations and either the ON, the cell membrane, or both.

Azides↗
PubMed1995

Exogenous nonphysiologic vs physiologic lipids. Divergent mechanisms for correction of permeability barrier dysfunction.

BACKGROUND AND DESIGN: Although barrier function requires cholesterol, free fatty acids, and ceramides, applications of one or two of these lipids to damaged skin impedes barrier recovery, while equimolar mixtures allow normal recovery. Both incomplete and complete mixtures appear to be internalized within the epidermal nucleated layers, followed by the secretion of abnormal vs normal lamellar body contents, respectively. We compared the ability of complete physiologic lipid mixtures vs a nonmetabolized hydrophobic lipid, petrolatum, to repair the barrier and the requirement for intracellular processing of these lipids within the epidermis. RESULTS: Neat petrolatum, which remains restricted to the stratum corneum, produces more rapid improvement in barrier function than the solvent-dispersed physiologic lipids, and its effects are not altered by coapplication of either monensin or brefeldin A (both from Sigma Chemical Co, St Louis, Mo), known inhibitors of exocytosis and organellogenesis, respectively. In contrast, the physiologic lipids enter the nucleated layers in substantial amounts and require longer to produce barrier recovery. Whereas monensin blocks their ability to facilitate barrier recovery, the physiologic lipids overcome brefeldin A-induced delays in barrier recovery, bypassing the subcellular site of brefeldin A blockade, normalizing both lamellar body contents and intercellular bilayers. CONCLUSIONS: While petrolatum remains restricted to the stratum corneum, physiologic lipid mixtures influence barrier recovery after transport to subjacent, nucleated layers, followed by internalization, apparent transport to the distal Golgi apparatus, and incorporation into nascent lamellar bodies.

Animals↗
PubMed1994

Antisense c-myc oligonucleotide cellular uptake and activity.

Previously described cell membrane transport mechanisms are unable to account completely for oligodeoxynucleotide cellular uptake. These charged macromolecules enter cells by an incompletely defined mechanism and downregulate gene expression in either the cytoplasm or nucleus. Thus, the goal of this research was to study the mechanism of phosphodiester oligonucleotide cellular uptake in Rauscher Red 5-1.5 erythroleukemia cells. An antisense c-myc oligodeoxynucleotide (21 bases) demonstrated biological activity in these cells using two types of proliferation assays and Northern blot analysis, and was internalized as visualized by confocal laser microscopy. Oligonucleotide uptake appeared to be a complex process consisting of surface binding and internalization. Cellular internalization accounted for up to 40% of total uptake and was partially dependent on both a trypsin-sensitive component and cellular energy. Uptake in these cells was nonspecific and did not appear to be due to receptor-mediated endocytosis. Therefore, because oligonucleotide cellular uptake in other cell types apparently involves an endocytic mechanism, the primary mechanism of oligonucleotide internalization may be cell line dependent.

Base Sequence↗
PubMed1994

Influence of altered serum cholesterol levels and fasting on cutaneous cholesterol synthesis.

Barrier perturbation stimulates epidermal cholesterol synthesis, which plays an important role in restoring barrier function. In the present study, we examined whether changes in serum cholesterol levels or nutrition regulate epidermal cholesterol synthesis in hairless mice. Serum cholesterol levels were lowered by 50% after injection with 4-aminopyrazolo (3,4-d) pyrimidine and were increased by 51% by feeding an atherogenic diet. In contrast to most other tissues, cholesterol synthesis in the epidermis and dermis was not inhibited by elevations or stimulated by decreases in serum cholesterol levels. Additionally, feeding a high-cholesterol diet did not decrease epidermal or dermal cholesterol synthesis. However, fasting significantly decreased both epidermal (38%) and dermal (34%) cholesterologenesis. Furthermore, barrier recovery after acetone disruption of the barrier was impaired in fasted animals. However, treatment with topical lipids did not restore barrier repair rate to normal, indicating that factors in addition to lipids are necessary to overcome the effects of fasting. These results demonstrate that cholesterol synthesis in the epidermis and dermis is regulated independently of changes in serum cholesterol levels.

Adenine↗
PubMed1994

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.

Animals↗
PubMed1992

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.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗