Antisense-treated Cells Research Articles

Urokinase Receptor Is Associated with the Components of the JAK1/STAT1 Signaling Pathway and Leads to Activation of This Pathway upon Receptor Clustering in the Human Kidney Epithelial Tumor Cell Line TCL-598

The urokinase-type plasminogen activator (uPA) binds to cells via a specific receptor attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Despite the lack of a transmembrane domain, the urokinase receptor (uPAR) is capable of transducing extracellular signals affecting growth, migration, and adhesion. Several Tyr kinases of the src family as well as beta1, beta2, and beta3 integrins were found to be associated with the uPAR. We found that in the human kidney epithelial line TCL-598, also components of the JAK1/STAT1 signal transduction pathway including gp130, are associated with uPAR as revealed by coimmunoprecipitation and are co-localized in caveolae. Upon clustering of uPA.uPAR complex by a monoclonal antibody, JAK1 associates with uPAR, which in turn leads to STAT1 phosphorylation, dimerization, specific binding to DNA, and gene activation. To prove the dependence of STAT1 activation on the uPAR, TCL-598 cells were treated with sense and antisense uPAR oligonucleotides. In antisense-treated cells in which uPAR expression was reduced to less then one third, activation of STAT1 by the clustering antibody was abolished while STAT1 activation by interferon-gamma was unaffected. Therefore, in this cell line, uPA.uPAR also utilizes the JAK1/STAT1 pathway for signaling, and gp130 might be the transmembrane adapter for this signal transduction pathway.

Antisense Inhibition of Group VI Ca2+-independent Phospholipase A2 Blocks Phospholipid Fatty Acid Remodeling in Murine P388D1 Macrophages

A major issue in lipid signaling relates to the role of particular phospholipase A2 isoforms in mediating receptor-triggered responses. This has been difficult to study because of the lack of isoform-specific inhibitors. Based on the use of the Group VI Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL), we previously suggested a role for the iPLA2 in mediating phospholipid fatty acid turnover (Balsinde, J., Bianco, I. D., Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92: 8527-8531). We have now further evaluated the role of the iPLA2 in phospholipid remodeling by using antisense RNA technology. We show herein that inhibition of iPLA2 expression by a specific antisense oligonucleotide decreases both the steady-state levels of lysophosphatidylcholine and the capacity of the cell to incorporate arachidonic acid into membrane phospholipids. These effects correlate with a decrease in both iPLA2 activity and protein in the antisense-treated cells. Collectively these data provide further evidence that the iPLA2 plays a major role in regulating phospholipid fatty acyl turnover in P388D1 macrophages. In stark contrast, experiments with activated cells confirmed that the iPLA2 does not play a significant role in receptor-coupled arachidonate mobilization in these cells, as manifested by the lack of an effect of the iPLA2 antisense oligonucleotide on PAF-stimulated arachidonate release.

Expression of adhesion molecules and functional stimulation in human neutrophils: modulation by GM-CSF and role of the Bcr gene.

Although devoid of proliferative capacity, polymorphonuclear neutrophils (PMN) express receptors for haemopoietic growth factors and need growth factors for survival and functional stimulation. This study showed that in vitro treatment of human PMN with GM-CSF for up to 48 h increases cell surface expression of the beta 2-integrin molecules CD11b/CD18 and CD11c/CD18 and of the receptor for the chemotactic peptide fMLP. Such modifications are usually expression of PMN activation. PMN treated with GM-CSF also displayed increased phagocytosis of latex particles and enhanced oxidative burst and superoxide anion release. Since integrins mediate PMN adhesion to endothelium, homotypic adhesion, chemotaxis/phagocytosis and the triggering of respiratory burst, our results suggested that functional stimulation of PMN persisted following prolonged exposure of PMN to growth factors and that it was not a temporary phenomenon which lasted only for the first 12-24 h of treatment. We also used oligonucleotides antisense to the Bcr gene mRNA to inhibit expression of the gene and evaluate its function in PMN, following the recent observation that PMN from Bcr-null mutant mice produced increased amounts of reactive oxygen metabolites upon activation. The antisense oligonucleotides had no effect on the parameters investigated. This may indicate that increased production of O2 by neutrophils in which the Bcr gene is not expressed requires either that gene expression is absent in the earlier stages of myeloid differentiation/maturation, so that when inhibition occurs in the terminally differentiated neutrophils their functional status is no longer influenced, or that the residual low-level expression of the gene which may be present in the antisense-treated cells is sufficient to provide a normal response to stimulation.

Expression of the Cystic Fibrosis Phenotype in a Renal Amphibian Epithelial Cell Line

Mutations in a Cl- channel (cystic fibrosis transmembrane conductance regulator or CFTR) are responsible for the cystic fibrosis (CF) phenotype. Increased Na+ transport rates are observed in CF airway epithelium, and recent studies suggest that this is due to an increase in Na+ channel open probability (Po). The Xenopus renal epithelial cell line, A6, expresses both cAMP-activated 8-picosiemen (pS) Cl- channels and amiloride-sensitive 4-pS Na+ channels, and provides a model system for examining the interactions of CFTR and epithelial Na+ channels. A6 cells express CFTR mRNA, as demonstrated by reverse transcriptase-polymerase chain reaction and partial sequence analysis. A phosphorothioate antisense oligonucleotide, complementary to the 5' end of the open reading frame of Xenopus CFTR, was used to inhibit functional expression of CFTR in A6 cells. Parallel studies utilized the corresponding sense oligonucleotide as a control. CFTR protein expression was markedly reduced in cells incubated with the antisense oligonucleotide. Incubation of A6 cells with the antisense oligonucleotide led to inhibition of forskolin-activated amiloride-insensitive short circuit current (Isc). After a 30-min exposure to 10 microM forskolin, 8-pS Cl- channel activity was detected in only 1 of 31 (3%) cell-attached patches on cells treated with antisense oligonucleotide, compared to 5 of 19 (26%) patches from control cells. A shift in the single-channel current-voltage relationship derived from antisense-treated cells was also consistent with a reduction in Cl- reabsorption. Both amiloride-sensitive Isc and Na+ channel Po were significantly increased in antisense-treated, forskolin-stimulated A6 cells, when compared with forskolin-stimulated controls. These data suggest that the regulation of Na+ channels by CFTR is not limited to respiratory epithelia and to epithelial cells in culture overexpressing CFTR and epithelial Na+ channels.

Inhibition of the Proliferative Cycle and Apoptotic Events in WiDr Cells after Down-Regulation of the Calcium-Binding Protein Calretinin Using Antisense Oligodeoxynucleotides

The colon adenocarcinoma cell line WiDr expresses the calcium-binding protein calretinin (CR). In order to deduce possible functions of calretinin in these cells we decreased its concentration by antisense techniques. Treatment of WiDr cells with phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) led to a drop in calretinin expression, as evidenced by immunohistochemical staining of WiDr cells and Western blot analysis of cytosolic cell extracts. The morphology of these epithelial cells changed from polygonal to spherical and they formed dense cell clusters. Cells displaying morphological alterations typical for apoptotic cells were observed after incubation with AS-ODNs, as evidenced by phase-contrast and electron microscopy. The mitotic rate of AS-ODN-treated cells dropped significantly, as demonstrated by mitotic labeling and time-lapse microcinematography. Furthermore, an accumulation of cells in phase G1 and a reduction of [3H]thymidine-labeled cells was observed in antisense-treated cells. The basal level of [Ca2+]iwas not influenced by the down-regulation of calretinin. WiDr cells incubated with the nonsense, reverse-sense, or with an oligodeoxynucleotide with a totally unrelated sequence did not show any significant differences when compared to control cells. We conclude that calretinin levels have an impact on the progression of the cell cycle of WiDr cells.

Inhibition of proliferation by L-myc antisense DNA for the translational initiation site in human small cell lung cancer: Dosaka-Akita H, Akie K, Hiroumi H, Kinoshita I, Kawakami Y, Murakami A. First Department of Medicine, Hokkaido University Sch. of Medicine, North 15, West 7, Kita-ku, Sopparo 060. Cancer Res 1995;55:1559–1564

We evaluated the antiproliferative effect of L-myc antisense DNA in NCI-H209, a human small cell lung cancer (SCLC) cell line overexpressing the L-myc gene. The synthetic DNA used in the present study was oligodeoxynucleoside phosphorothioate, which showed rapid incorporation into NCI-H209 cells and localized mainly in the cell nucleus and weakly in the cytoplasm. The exposure of this cell line to L-myc antisense DNA covering the translational initiation site of L-myc proteins inhibited the cell proliferation in a dose-dependent sequence-specific manner. Furthermore, the growth inhibition by this antisense DNA was correlated with the level of L-myc expression in three SCLC cell lines, NCI-H209, NCI-H510, and NCI-H82. In Western blot analysis, expression of the L-myc proteins was down-regulated in the antisense-treated cells compared with control-treated cells in NCI-H209. Together with unique characteristics of the L-myc gene, including: (a) a frequently amplified and overexpressed state in SCLC; and (b) very restricted and low-level expression in human adult tissues, the present data indicate that L-myc is a good candidate for the target gene for antisense DNA therapy based on molecular biological diagnosis in SCLC.

Antisense oligodeoxynucleotides to the blk tyrosine kinase prevent anti-mu-chain-mediated growth inhibition and apoptosis in a B-cell lymphoma.

Crosslinking of membrane immunoglobulin (mIg) receptors by anti-Ig causes growth inhibition and subsequent cell death due to apoptosis in a murine B-cell lymphoma model. The earliest signal transduction via mIg has recently been shown to be dependent on the activation of one or more protein tyrosine kinases (PTKs). In this study, we utilized the CH31 lymphoma, which is extremely sensitive to growth inhibition by anti-Ig, to examine the role of PTKs in cell cycle arrest. This cell line expresses multiple PTKs, whose activities are stimulated by crosslinking mIg. To determine whether PTK activity is essential for the inhibition of cell growth, we exposed CH31 cells to antisense oligodeoxynucleotides for the blk PTK prior to the growth inhibition assay. We found that exposure of CH31 cells to blk antisense effectively prevented anti-mu-chain-mediated growth inhibition and subsequent apoptosis. Corresponding blk sense or antisense oligonucleotides for other PTKs had no protective effect against anti-mu. Moreover, antisense blk oligonucleotides had no effect on transforming growth factor beta-mediated growth arrest and apoptosis. Further experiments showed significantly reduced endogenous p55blk in blk antisense-treated cells. In addition, anti-mu stimulation of antisense-treated cells failed to induce any detectable increase in kinase activity of p55blk, a result suggesting the uncoupling of blk proteins from normal signal pathways that are essential for growth inhibition. These results implicate a role of blk kinase in anti-mu-mediated pathway to cell cycle arrest.

Viral interleukin 10 is critical for the induction of B cell growth transformation by Epstein-Barr virus.

We have used an efficient cDNA subtraction library procedure to identify newly induced genes in human B lymphocytes infected for 6 h with Epstein-Barr virus (EBV). Among the genes identified by automated sequencing of a random subset of clones from this library, one coded the EBV BCRF1 open reading frame, which specifies the viral interleukin 10 gene (vIL-10). This molecule is highly homologous to human (h)IL-10 and was previously thought to represent a "late" viral gene expressed only during the lytic phase of virus replication. Using gene amplification by reverse transcriptase polymerase chain reaction of B cell RNA obtained at varying times after infection, we detected vIL-10 expression within a few hours of EBV infection, followed, 20-30 h later by expression of hIL-10. Expression of both genes continued beyond the initial transformation phase (5-10 d) and was present in all transformed cell lines tested. When added at the time of viral infection, antisense (but not sense) oligonucleotides for vIL-10 mRNA (cytosolic half-life, approximately 6 h) prevented subsequent B cell transformation. The antisense effect was highly specific, leaving the expression levels of other transformation-related genes intact. Addition of exogenous (h)IL-10 rescued the transformation process in antisense-treated cells. Our observations establish vIL-10 as a new latency gene with a directly transformation-prerequisite function.

Antiproliferative Effect of DNA Polymerase α Antisense Oligodeoxynucleotides on Breast Cancer Cells

Antisense oligonucleotides appear to offer considerable promise as sequence-specific inhibitors of gene expression. Different cellular targets for oligodeoxynucleotides with oncologic interest have been identified such as oncogenes, growth factors, and cell cycle-related genes. DNA polymerase α (polα) plays a relevant role in DNA synthesis and cell proliferation. Polα gene expression is constitutive throughout the cell cycle and its mRNA content and activity are related to the growth rate and neoplastic phenotype. The effects of a 18-mer polα antisense oligomer on the proliferation of the MDA-MB 231 breast cancer cell line have been investigated. After 48 h in culture with oligomers (10 μ M), about 50% growth inhibition was observed in antisense-treated cells, as evaluated by 3-(4, 5-dimethythiazol-2yl)-2, 5-diphenyltetrazolium bromide assay and cell count. [ 3H]Thymidine incorporation exhibited a 90% inhibition of DNA synthesis associated to 64% accumulation of cells at the G1-S border of the cycle as by flow cytometry, at 24 h. Northern hybridization and SDS-PAGE of immunoprecipitated MDA-MB 231 cell lysates revealed a decreased expression of polα mRNA and a reduction of the 180-kDa polypeptide, respectively. Collectively, the data further confirm the relevance of polα in the replicative cycle, as well as strengthen the potentiality of the antisense strategy for the control of gene expression and cell growth.

Growth Inhibition of Ovarian Cancer Cells Induced by Antisense IL-6 Oligonucleotides

In previous work, we saw that interleukin-6 (IL-6), a multifunctional cytokine, is produced by epithelial ovarian cancer cells and that ovarian cancer cells express the IL-6 receptor. Here, we examined the possibility that IL-6 acts as an autocrine growth factor for ovarian cancer cells. Inhibition of IL-6 gene expression by exposure to IL-6 antisense oligonucleotides resulted in greatly decreased cellular proliferation. Exposure of ovarian cancer cell lines (CAOV-3, OVCAR-3, and OC-436), to 1-5 μM of a 15-base single-stranded antisense IL-6 oligodeoxynucleotide, specific for a sequence in the second coding exon of the IL-6 gene, resulted in decreased IL-6 production and a >80-85% inhibition of cellular proliferation. However, the addition of exogenous IL-6 failed to restore the proliferation of the antisense-treated cells. Antibodies to IL-6 did not consistently inhibit cell growth nor did rIL-6 enhance precursor frequency in a limiting dilution analysis. These results suggest that IL-6 does not directly induce the proliferation of ovarian cancer cells although endogenous IL-6 production is needed for optimal cell growth. As the majority of epithelial ovarian cancers produce IL-6, the direct specific inhibition of IL-6 gene expression is of potential therapeutic value.

Suppression of kinesin expression in cultured hippocampal neurons using antisense oligonucleotides.

Kinesin, a microtubule-based force-generating molecule, is thought to translocate organelles along microtubules. To examine the function of kinesin in neurons, we sought to suppress kinesin heavy chain (KHC) expression in cultured hippocampal neurons using antisense oligonucleotides and study the phenotype of these KHC "null" cells. Two different antisense oligonucleotides complementary to the KHC sequence reduced the protein levels of the heavy chain by greater than 95% within 24 h after application and produced identical phenotypes. After inhibition of KHC expression for 24 or 48 h, neurons extended an array of neurites often with one neurite longer than the others; however, the length of all these neurites was significantly reduced. Inhibition of KHC expression also altered the distribution of GAP-43 and synapsin I, two proteins thought to be transported in association with membranous organelles. These proteins, which are normally localized at the tips of growing neurites, were confined to the cell body in antisense-treated cells. Treatment of the cells with the corresponding sense oligonucleotides affected neither the distribution of GAP-43 and synapsin I, nor the length of neurites. A full recovery of neurite length occurred after removal of the antisense oligonucleotides from the medium. These data indicate that KHC plays a role in the anterograde translocation of vesicles containing GAP-43 and synapsin I. A deficiency in vesicle delivery may also explain the inhibition of neurite outgrowth. Despite the inhibition of KHC and the failure of GAP-43 and synapsin I to move out of the cell body, hippocampal neurons can extend processes and acquire as asymmetric morphology.

Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells.

We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment (3.9 or 1.3 microM) for 24 hr virtually abolished Cl- transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl- transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl- permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl- permeability.