ISSN:1050-5261    

DOI:10.1089/ard.1995.5.241

年代:1995

数据来源: MAL

摘要:

Replication-defective adenovirusp259Acaused a 400-fold increase in the sequence-specific antiherpetic activity of oligo(nucleoside methylphosphonate) (ONMP) IE4,5SA. Herpes simplex virus type 1 (HSV-1) growth was not inhibited in cells exposed top259Ain the absence of IE4,5SA or in cells cotreated with I E4,5SA and heated (10 minutes, 90°C)p259Avirus. Fluorescent microscopy of Vero cells treated with BODIPY-conjugated IE4,5SA revealed intracellular localization within endocytic-like vesicles with minimal cytoplasmic and intranuclear distribution. Diffuse staining over the entire cell was observed in cell cotreated with the BODIPY-conjugated IE4,5SA andp259Avirus. This effect was not observed in cells cotreated with the BODIPY-conjugated ONMP and heatedp259Avirus. We interpret these findings to indicate thatp259Aaugments IE4,5SA antiherpetic activity presumably via its ability to increase ONMP uptake and release from endocytic-like vesicles

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.243

年代:1995

数据来源: MAL

摘要:

The cellular uptake of oligodeoxyribonucleoside methylphosphonates has been evaluated using three radiolabeled oligomers. Oligomers I and II ([3H]-T8and [3H]-T16, respectively) are nonionic methylphosphonate oligomers labeled with tritium on the phosphonate internucleotide linkage. EDA-III contains a single phosphodiester linkage, a [32P]-label and an ethylenediamine conjugate at the [32P]-5′-end. All three oligomers are stable in cells. At a 1 μM concentration, oligomer I is not taken up by human erythrocytes. The octanol/DPBS partition coefficients for oligomers I and II (1.5 x 10-4and 4.2 x 10-4, respectively) further indicate that these molecules should not diffuse across cell membranes at appreciable rates. Oligomer I is taken up by HL-60 cells, although at a slower rate than the uptake of the fluid-phase marker sucrose. The cell-associated levels of oligomer II in K-562 cells following incubation of cells with the oligomer for 2 days is independent of concentration and nonsaturable, suggesting a mechanism of uptake independent of receptor. Finally, the initial uptake rate of EDA-III in mouse L cells is greater than the uptake of two oligodeoxyribonucleotides (T8,T16), reaching a plateau after 3 hours incubation with cells. These observations should aid in the elucidation of the mechanism by which this class of antisense agents enters the intracellular environme

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.251

年代:1995

数据来源: MAL

摘要:

To evaluate the effect of synthetic DNA oligomers on regulation of bacterial genesin vivo, we tested 63 oligomers of variable length and chemistry for their ability to selectively suppress light production in the bioluminescent marine organism,Vibrio fischeri. Phosphodiester, phosphorothioate, and mixed backbone oligomers were designed to beluxgene targeted or nontargeted (negative) controls. Although significant suppression of luminescence was observed, most notably with the phosphorothioate oligomers, there was no correlation between inhibitory activity and oligomer sequence. The phosphorothioate oligomer that was most potent for inhibition of luminescence in bacterial culture had no effect on the activity of purified luciferase. Mechanisms other than sequence-specific inhibition of gene expression or direct interaction with luciferase are discussed.

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.261

年代:1995

数据来源: MAL

摘要:

In our efforts to investigate the biologic role of Ha-rasoncogenes in human melanoma by Ha-rasphosphorothioate antisense oligonucleotides, we observed that antisense, sense, and scrambled control oligonucleotides at a concentration of 10 μM all similarly and strongly inhibited growth of our human melanoma target cell line SK-2in vitrobut without specific decrease of the target protein. Cell numbers with respect to the untreated control were reduced by 84% ± 4.2% (ISD), 82.9% ± 3.6%, and 84% ± 3%, respectively.In vivostudies in a SCID-hu mouse model confirmed these findings. Both antisense and sense control oligonucleotides administered through osmotic pumps significantly (p<0.006) reduced the mean tumor weight (1.5 g ± 0.4 g and 1.8 g ± 0.8 g, respectively) in comparison with saline-treated (5.7 g ± 0.7 g) or untreated control animals (5.8 g ± 1.0 g). The vascularity of oligonucleotide-treated tumors was greatly reduced. Clinical signs of oligonucleotide-related toxicity were not observed, and there was no evidence of histopathologic alterations in a variety of mouse tissues. We could demonstrate that the antimelanoma effects can be abrogatedin vitroby adding basic fibroblast growth factor (bFGF). In the context of the importance of bFGF in melanocyte biology and angiogenesis, we argue in favor of an interaction between polyanionic phosphorothioate oligonucleotides and bFGF in our melanoma system. These findings stress the notion that phosphorothioate oligonucleotides may be promising antineoplastic lead compounds capable of employing antitumor effects by mechanisms other than specific inhibition of gene exp

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.271

年代:1995

数据来源: MAL

摘要:

To clarify the renal disposition characteristics of oligonucleotides at the organ level, the renal handling of model end-capped oligonucleotides, 3′- methoxyethylamine 5′-biotin-decathymidylic acid containing phosphoramidate modifications at 3′- and 5′-terminal internucleoside linkages (T10) and its phosphorothioate (Ts10), were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous outflow and urinary excretion patterns and tissue accumulation of radiolabeled oligonucleotides were evaluated under filtering or nonfiltering conditions. No significant binding to bovine serum albumin (BSA) in the perfusate was observed for T10, whereas more than 90% of Ts10bound to BSA. The steady-state distribution volume of T10calculated from the venous outflow pattern was larger than that of inulin, which corresponds to the extracellular volume of the kidney, whereas the distribution volume of Ts10was larger than that of BSA (the intravascular volume). These results suggested their interaction with the vascular wall. Rapid urinary excretion was observed for T10, similar to inulin used as a marker of golmerular filtration rate. On the other hand, urinary excretion of Ts10was greatly restricted due to its high binding ability (>90%) to BSA in the perfusate. A significant amount of T10and Ts10was accumulated in the kidney (T10,1.8% of injected dose; Ts10, 1.3%) compared with inulin (0.2%) and BSA (<0.1%). The accumulation of these oligonucleotides was ascribed to both tubular reabsorption and uptake from the capillary side. In addition, the uptake of T10from the capillary side was significantly inhibited by simultaneous injection of dextran sulfate, suggesting that the oligonucleotide was taken up as an anionic molecule. These findings will be useful information for the development of delivery systems for antisense oligonucl

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.279

年代:1995

数据来源: MAL

摘要:

We have successfully used antisense RNA to inhibit replication of the mouse hepatitis virus (MHV) in a cell culture system. MHV is a single-stranded RNA virus of positive polarity. Mouse L2 cells were stably transfected with an antisense construct that targets regions of genes 5 and 6 of the virus. High levels of expression from this construct, which is under control of the human elongation factor 1α promoter, were found. After infection of the antisense cell lines with MHV, replication of the virus was significantly reduced compared with control cells. In a viral plaque assay, smaller plaques were found in the antisense cell lines. In addition, up to a 92% inhibition in the number of viral particles produced in one antisense cell line could be seen. This inhibitory effect decreased at longer (>16 hour) infection times. It was possible to both increase the amount of inhibition and prolong the inhibitory effect by reducing the multiplicity of infection. Our results suggest that antisense RNA may be an effective tool to slow down progression of MHV infection in mice

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.289

年代:1995

数据来源: MAL

摘要:

A genetic system for the analysis of antisense and ribozyme mechanisms is a much needed experimental tool, and yeast represent a favorable organism on which to base such a system. We have shown previously that the fission yeastSchizosaccharomyces pombehas potential to satisfy the requirements of such a system. This report describes experiments designed to determine if antisense and ribozyme RNA-mediated gene suppression will be generally applicable to other genes inS. pombe. Antisense and ribozyme RNAs designed to suppress theade6gene were expressed at high levels from episomal expression vectors. Theade6gene was chosen as a target as mutations within the gene confer adenine auxotrophy and a red colony phenotype, and it was expected that antisense or ribozyme RNA-mediated mutant phenocopies would exhibit the same readily detectable phenotype. No phenotypic indication ofade6suppression was detected in transformed yeast, andade6target mRNA was analyzed by primer extension and Northern analysis. Initially, conflicting results were obtained from these techniques, which were determined to be due to duplex formation between antisense and target RNAin vitro. No detectable reduction in theade6mRNA levels was found, and it was concluded that the gene was not suppressed by the antisense or ribozyme RNAs tested. These results confirm that inS. pombeas with other organisms, the susceptibility of genes to RNA-mediated suppression may be gene specific and that design of antisense and ribozyme genes will be an empirical process.

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.295

年代:1995

数据来源: MAL

摘要:

The association of urokinase-type plasminogen activator (uPA) with its receptor (uPAR) influences various biologic functions, including cell migration, angiogenesis, differentiation, and wound healing. Expression of uPAR at the mesothelial surface could, therefore, influence cellular responses in the pleural space. We found that a line of cultured human mesothelial cells (MeT5A) expressed specific and saturable binding sites for uPA that increased on stimulation with PMA. Ligand blotting studies showed that the mesothelial receptor is a 50 kD protein similar to that in other cell lines. Binding of active and intact, but not amino terminal or low molecular weight fragment, uPA to mesothelial cells enhanced DNA synthesis and cell proliferation, and antibodies against either the active site of uPA or uPAR abrogated this effect. We reasoned that regulation of uPAR expression could control uPA-induced mitogenesis and tested this hypothesis with antisense oligonucleotides complementary to uPAR mRNA. Phosphorothioate-modified antisense oligonucleotides inhibited uPA-mediated mesothelial cell proliferation in a concentration-dependent manner. These effects were associated with decreased binding of125I-uPA and reduced expression of the uPAR gene product. The results indicate that uPAR is involved in signal transduction pathways that control uPA-mediated mesothelial cell proliferation, a process implicated in the pathogenesis of mesothelial inflammation and pleural neoplasia. Antisense oligonucleotides to uPAR suppress mesothelial cell mitogenesisin vitroand offer a potential means of regulating the processin vivo.

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.307

年代:1995

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1995.5.315

年代:1995

数据来源: MAL