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At least 73 records · Page 4Linked to original sources
PubMed2004

Inhibition of tumour necrosis factor-alpha by antisense targeting produces immunophenotypical and morphological changes in injury-activated microglia and macrophages.

Microglia respond in a stereotypical pattern to a diverse array of pathological states. These changes are coupled to morphological and immunophenotypical alterations and the release of a variety of reactive species, trophic factors and cytokines that modify both microglia and their cellular environment. We examined whether a microglial-produced cytokine, tumour necrosis factor-alpha (TNF-alpha), was involved in the maintenance of microglial activation after spinal cord injury by selective inhibition using TNF-alpha antisense deoxyoligonucleotides (ASOs). Microglia and macrophages harvested from 3 d post-contused rat spinal cord were large and rounded (86.3 +/- 9.6%). They were GSA-IB4-positive (GSA-IB4(+)) (Griffonia simplicifolia lectin, microglia specific; 94.8 +/- 5.1%), strongly OX-42 positive (raised against a type 3 complement/integrin receptor, CD11b; 78.9 +/- 9.1%), ED-1 positive (a lysosomal marker shown to correlate well with immune cell activation; 97.2 +/- 2.6%) and IIA positive (antibody recognizes major histocompatibility complex II; 57.2 +/- 5.6%), indicative of fully activated cells, for up to 48 h after plating. These cells also secreted significant amounts of TNF-alpha (up to 436 pg/microg total protein, 16 h). Fluoroscein isothiocyanate-labelled TNF-alpha ASOs (5, 50 and 200 nm) added to the culture medium were taken up very efficiently into the cells (> 90% cells) and significantly reduced TNF-alpha production by up to 92% (26.5 pg/microg total protein, 16 h, 200 nm TNF-alpha ASOs). Furthermore, few of the treated cells at this time were round (5.4 +/- 2.7%), having become predominantly spindle shaped (74.9 +/- 6.3%) or stellate (21.4 +/- 2.7%); immunophenotypically, although all of them remained GSA-IB4 positive (91.6 +/- 6.2%), many were weakly OX-42 positive and few expressed either ED-1 (12.9 +/- 2.5%) or IIA (19.8 +/- 7.4%). Thus, the secretion of TNF-alpha early in spinal cord injury may be involved in autoactivating microglia/macrophages. However, at the peak of microglial activation after injury, the activation state of microglia/macrophages is not stable and this process may still be reversible by blocking TNF-alpha.

Animals↗
PubMed1997

Antisense targeting of basic fibroblast growth factor and fibroblast growth factor receptor-1 in human melanomas blocks intratumoral angiogenesis and tumor growth.

Unlike normal melanocytes, primary and metastatic human melanomas express high levels of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1) messenger RNA, and expression of these genes is essential in sustaining the proliferation of malignant melanomas in vitro. To determine whether bFGF and FGFR-1 are also required for tumor formation in these cells, liposome-mediated gene transfer was used to deliver episomal vectors containing antisense-oriented bFGF or FGFR-1 cDNAs into human melanomas, grown as subcutaneous tumors in nude mice. The growth of tumors injected with these constructs was completely arrested or the tumors regressed as a result of blocked intratumoral angiogenesis and subsequent necrosis. Thus, inhibition of bFGF/FGFR-1-mediated signaling may open a new avenue for the treatment of advanced-stage melanomas.

Animals↗
PubMed2006

Antisense targeting of CXXC finger protein 1 inhibits genomic cytosine methylation and primitive hematopoiesis in zebrafish.

CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is a component of the Set1 histone methyltransferase complex. Mice lacking CFP1 suffer a peri-implantation lethal phenotype, and CFP1-deficient embryonic stem cells are viable but unable to differentiate and exhibit a 60-80% decrease in genomic cytosine methylation. A zebrafish homolog of CFP1 has been identified, is approximately 70% similar to murine CFP1, and is widely expressed during development. Zebrafish embryos treated with a zCFP1 antisense morpholino oligonucleotide had little or no circulating red blood cells and exhibited abnormal yolk sac morphology at 48 h post-fertilization. Many of the antisense-treated zebrafish also exhibited cardiac edema, and 14% were dead at 24 h post-fertilization. Morphant zebrafish also exhibited elevated levels of apoptosis, particularly in the intermediate cell mass, the site of primitive erythropoiesis, as well as aberrations in vascular development. Genomic DNA isolated from morphant embryos exhibited a 60% reduction of global genomic cytosine methylation. A similar phenotype was observed with an independent zCFP1 antisense morpholino oligonucleotide, but not following injection of an unrelated control oligonucleotide. The morphant phenotype was rescued when mRNA encoding murine CFP1 was co-injected with the antisense oligonucleotide. Genomic data base analysis reveals the presence of a second version of zebrafish CFP1 (zCFP1b). However, the morphant phenotype observed following specific depletion of zCFP1 indicates that these related genes have nonredundant functions controlling normal zebrafish hematopoiesis and epigenetic regulation. These findings establish the importance of CFP1 during postgastrulation development.

Amino Acid Motifs↗
PubMed1997

Antisense targeting of E6AP elevates p53 in HPV-infected cells but not in normal cells.

Invasive cervical cancer is very highly correlated with the presence of high-risk human papillomavirus (HPV) types 16 and 18. Two viral proteins, E6 and E7, act in concert to subvert growth control of infected cells by inactivating the tumor suppressor proteins, p53 and Rb, respectively. E6 is thought to abrogate p53 function by stimulating its degradation via ubiquitin-mediated proteolysis in a reaction requiring E6AP (E6-Associated Protein). Here we evaluate the in vivo role of E6AP in p53 degradation in normal and HPV-infected cell types using antisense phosphorothioate oligodeoxynucleotides (S-ODNs). This study shows that reduction of E6AP in vivo in high-risk HPV-infected cells leads to an elevation of p53, confirming the function of E6AP predicted by in vitro experiments. Further, we demonstrate that reduction of E6AP in normal cells has no effect on p53 levels, indicative of an E6AP-indpendent mechanism for p53 degradation. These experiments show that inhibition of intermediate proteins in the ubiquitin-mediated proteolysis pathway (ubiquitin-conjugating enzymes or associated recognition proteins) can result in specific inhibition of substrate degradation. We propose that modulation of p53 levels by elimination of E6AP function may have therapeutic potential for cervical cancer.

Apoptosis↗
PubMed2003

Antisense targeting protein kinase A type I as a drug for integrated strategies of cancer therapy.

We have studied the role of protein kinase A (PKA) and its type I isoform (PKAI) in the transduction of mitogenic signaling, apoptosis, and angiogenesis. We have contributed to the development of selective inhibitors of PKAI, including a hybrid DNA/RNA mixed backbone oligonucleotide (AS-PKAI). We, and others, have demonstrated that AS-PKAI has a cooperative antitumor effect with a selected class of cytotoxic drugs and with radiotherapy in vitro and in vivo and that these effects can also be obtained following oral adinistration. Previously, we developed a series of therapeutic models based on the pleiotropic role played by PKAI in cell proliferation, apoptosis, and angiogenesis. On the basis of our former demonstration of functional and structural interactions of PKAI and the activated epidermal growth factor receptor (EGFR), we have shown that the combined blockade of both signaling molecules by AS-PKAI and either the monoclonal antibody C225 (erbitux) or the small molecule ZD1839 (gefitinib), results in a marked cooperative antitumor effect in a variety of human tumor models. A further cooperative antitumor effect can be obtained when AS-PKAI is used in combination with both EGFR inhibitors and either cytotoxic drugs or radiotherapy. The antitumor activity is associated with inhibition of growth factors and angiogenic factors production and to induction of apoptosis. In light of the recently demonstrated role of PKAI on the bcl-2-dependent apoptotic pathway, we have recently shown a synergistic antitumor, antiangiogenic, and proapoptotic effect of AS-PKAI in combination with antisense bcl-2 (oblimersen) or with a bispecific bcl-2/bcl-xL second generation antisense. A connection between COX-2, EGFR and PKAI was established, and we demonstrated that the combination of AS-PKAI with gefitinib and a COX-2 inhibitor, all adminstered orally, can result in a potent antitumor and antiangiogenic activity. These studies support the development of AS-PKAI as a novel anticancer agent and suggest its potentially relevant role when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

Animals↗
PubMed1995

Reversal of drug sensitivity in MDR subline of P388 leukemia by gene-targeted antisense oligonucleotide.

We attempted to reverse multidrug resistance (MDR) by treatment with 25-mer antisense phosphorothioate oligonucleotide. The phosphorothioate analogs, the sequences of which are sense or antisense to the initiation codon of mouse mdr1 mRNA, were tested against murine leukemic P388/S and adriamycin-resistant P388/ADR cell lines. A weak inhibitory effect on the growth of P388/S and P388/ADR cells was observed at a sense and antisense oligonucleotide concentration of 30 microM. Using the monoclonal antibody to P-glycoprotein and a flow cytometry technique, we showed that the level of expression of P-glycoprotein in P388/ADR cells treated with antisense oligonucleotide was lower than when treated with sense oligonucleotide. The antisense oligonucleotide potentiated the growth-inhibitory effect of vinblastine on P388/ADR cells, whereas sense oligonucleotide did not. This was accompanied by an increase in vinblastine retention in the cells. The reversal of the resistance by antisense oligonucleotide was increased by the combination with 1 microM verapamil. These results suggest that the antisense oligonucleotide and low dose verapamil may be useful in circumventing the resistance to anticancer drugs of MDR tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Oncogene-targeted antisense oligodeoxynucleotides combined with chemotherapy or immunotherapy: a new approach for tumor treatment?

Synthetic oligodeoxynucleotides (antisenses) complementary to bcr/abl breakpoint junction transcript on Philadelphia chromosome, or c-myb protooncogene inhibit partially the proliferation of Philadelphia positive leukemic cells (antisenses against bcr/abl and c-myb) and other tumor cells (antisenses against c-myb). This phenomenon is accompanied by specific downregulation of mRNA level of the particular gene. To develop a more effective procedure of tumor treatment the combination of low dose of cytostatic and bcr/abl or c-myb antisenses against Philadelphia chromosome positive cell line BV173, and the combination of anti-tumor cytotoxic T lymphocytes (CTL) and c-myb antisenses against melanoma cell line MM-28, were tested in vitro. Our results indicate that the combinations of conventional chemotherapeutic agent and antisense against bcr/abl or c-myb or tumor specific CTL and antisense against c-myb, are highly effective in killing of tumor cells and sparing normal cells. This creates the possibility to develop a more selective and effective treatment of neoplasia.

Bone Marrow Cells↗
PubMed2005

Schizophyllan-folate conjugate as a new non-cytotoxic and cancer-targeted antisense carrier.

Schizophyllan having folate-appendages was synthesized from native schizophyllan through NaIO(4)-oxidation and the subsequent reductive amination in aqueous ammonia followed by amido-coupling with folic acid. The resulting folate-appended schizophyllan can form stable complex with poly(dA), show specific affinity toward folate binding protein, and mediate effective antisense activity in cancer cells.

Base Sequence↗
PubMed2004

Schizophyllans carrying oligosaccharide appendages as potential candidates for cell-targeted antisense carrier.

Schizophyllans carrying beta-lactoside and alpha-mannoside appendages were prepared from native schizophyllan through NaIO4 oxidation followed by reductive amination using aminoethyl-beta-lactoside and alpha-mannoside, respectively. The resulting schizophyllans form stable macromolecular complexes with polynucleotides, such as poly(C) and poly(dA). Specific affinity between these macromolecular complexes and saccharide-binding proteins was demonstrated by surface plasmon resonance and agarose gel staining assays. beta-lactoside-appended schizophyllan enhanced an antisense activity in hepatocytes which express lactoside-binding proteins on their cell surfaces.

Cell Count↗
PubMed2003

Specific iNOS-targeted antisense knockdown in endothelial cells.

The inhibition of inducible nitric oxide synthase (iNOS) expression via antisense oligonucleotides (AS-ODN) may represent a highly specific tool. Endothelial cells (EC) represent prime candidate cells for in vivo application, and we therefore aimed at optimizing this technique for effectiveness and specificity in primary nontransformed rat EC. EC or L929 fibroblasts were incubated with iNOS-specific ODN optimizing all experimental steps. We find that ODN uptake, as analyzed by fluorescence microscopy and labeled ODN, was absolutely dependent on vehicle presence, and among the vehicles tested, Lipofectin displayed negligible toxicity and good uptake. In addition, omission of serum was also essential, a factor that might limit its use in vivo. Moreover, intranuclear accumulation of AS-ODN appeared crucial for successive inhibition. The impact of ODN on iNOS mRNA, protein, and enzyme activity was specific and resulted in >95% inhibition of protein formation. In conclusion, in this article we provide a protocol for an optimized AS-mediated knockdown, representing a specific and efficient instrument for blocking of iNOS formation and allowing for studying the impact of iNOS expression on endothelial function. We also expose application problems of this technique when working in inflammatory conditions.

Animals↗
PubMed2005

In vivo potentialities of EWS-Fli-1 targeted antisense oligonucleotides-nanospheres complexes.

The EWS/FLI-1 fusion gene, resulting from a t(11;22) translocation, plays a key role in the pathogenesis of Ewing sarcoma. Previously, we have shown that antisense oligonucleotides designed against EWS-Fli-1 inhibited tumor growth in nude mice provided they were delivered intratumorally by nanocapsules or by CTAB-coated nanospheres. In this study, we have used two types of nanospheres (designated as type 1 and type 2 nanospheres) stabilized with chitosan for both intratumoral and systemic administration of oligonucleotides. Inhibition of the tumor growth in vivo was found to be dependent on the carrier type as well as on antisense oligonucleotide modification. Indeed, whereas both types of nanospheres were efficient in reducing tumor growth after intratumoral injection, we have obtained only with type 2 nanospheres an antitumoral effect after intravenous injection in a preliminary experiment. Additionally, the anticancer efficacy of a localized modification of the EWS-Fli-1 phosphodiester/phosphorothioate chimeric antisense oligonucleotide was demonstrated. In cell culture the oligonucleotides inhibit cell growth by their antisense activity. Further investigations are needed in vivo to learn the mechanism of action of the complexes.

Animals↗
PubMed2000

Modulation of multidrug resistance in human leukemia cells with mdr1-targeted antisense oligonucleotides using variable treatment schedules.

OBJECTIVE: The purpose of the current study was to characterize the effect of chimeric AS-ODNs encapsulated with cationic lipids on MDR in human leukemia cells and to determine if this modification of the ODN alone or in combination with the cationic lipid might offer advantages over classical ODN treatment with free unmodulated or phosphorothiolated AS-ODNs. Furthermore, we extended the antisense method to the use of AS-ODNs in the parental drug-sensitive leukemia cells which express mdr1-mRNA at a relative low level and lack P170 expression to evaluate the effectiveness of prophylactic AS-ODN treatment. METHODS: The effect of a 4-day AS-ODN treatment in drug-resistant human leukemia cells which exhibit the classic MDR phenotype at a moderate level was examined. Twenty-four hours after the last ODN administration the cells were analyzed for mdr1-mRNA (quantitative RT-PCR) and P170 expression (FCM), for R123 accumulation/efflux capacity (FCM) and for sensitivity to vincristine (MTT). In the parental drug-sensitive CCRF-CEM cells the mdr1-mRNA expression was assessed 24, 48 and 72 h after AS-ODN treatment administered as free phosphorothioate or conjugated with DMRIE-C. RESULTS: Cationic lipids produced a clear increase in cellular ODN uptake but also caused an increase in variability of uptake rates (30% vs. 10% variability after free phosphorothioates). Both AS-ODNs inhibit P170 expression whereby the antisense effect of the chimeric ODN seems to be stronger compared to the phosphorothioate (30% vs. 22% MRK16 staining). Consistent with the inhibition of P170 expression, an increased sensitivity to vincristine was observed. In parental drug-sensitive cells, AS-ODN treatment caused nearly complete inhibition of mdr1-mRNA expression (5% of control). CONCLUSION: The data demonstrate that it is nearly impossible to achieve a complete reversal of the MDR phenotype in drug-resistant cells using AS-ODNs. A more promising approach seems to be the prophylactic treatment with AS-ODNs.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Antisense oligonucleotide targeting of Raf-1: importance of raf-1 mRNA expression levels and raf-1-dependent signaling in determining growth response in ovarian cancer.

PURPOSE: We sought to identify determinants of growth response to the Raf-1-targeted antisense oligonucleotide (ASO; ISIS 5132) using a large panel of ovarian cancer cell lines. EXPERIMENTAL DESIGN: First-(ISIS 5132) and second-generation (ISIS 13650) anti-Raf 1 ASOs were compared with control oligonucleotides. Growth was assessed by cell counts; apoptosis was assessed by poly(ADP-ribose) polymerase cleavage; and cell cycle analysis was assessed by flow cytometry. Protein expression was detected by Western blot analysis, and mRNA expression was detected by quantitative reverse transcription-PCR. Raf-1 kinase activity was detected by anti-Raf-1 immunoprecipitation, followed by myelin basic protein phosphorylation. RESULTS: A panel of 15 ovarian cancer cell lines was used to model a range of growth responses to ASOs targeting Raf-1 mRNA. Growth inhibition varied from 10% to >90% inhibition. Growth inhibition was associated with increased apoptosis and accumulation of cells in the G(2)-M and S phases of the cell cycle. Growth response was not related to level of Raf-1 protein expression, Raf-1 kinase activity, intracellular ASO uptake, or degree of Raf-1 protein inhibition. However, ASO growth response was associated with a high proportion of Raf-1 mRNA [relative to total (i.e., Raf-1 + A-Raf + B-Raf) Raf mRNA] and significantly higher Raf-1 kinase activity induction following growth factor (transforming growth factor alpha) stimulation in the cell lines consistent with dependency of these cell lines on Raf-1. CONCLUSIONS: These data indicate that ovarian cancers demonstrate differential sensitivity to ASOs targeted against Raf-1, and target expression levels and degree of utilization of Raf-1 signaling are implicated. Clinically sensitive tumors could feasibly be identified.

Base Sequence↗
PubMed1998

Tetranucleotide GGGA motif in primary RNA transcripts. Novel target site for antisense design.

Selecting effective antisense target sites on a given mRNA molecule constitutes a major problem in antisense therapeutics. By trial-and-error, only 1 in 18 (6%) of antisense oligonucleotides designed to target the primary RNA transcript of tumor necrosis factor-alpha (TNF-alpha) strongly inhibited TNF-alpha synthesis. Subsequent studies showed that the area in RNA targeted by antisense oligonucleotides could be moved effectively 10-15 bases in either direction from the original area. We observed that only molecules that incorporated a tetranucleotide motif TCCC (complementary to GGGA on RNA) yielded potent antisense oligonucleotides against TNF-alpha. A comprehensive literature survey showed that this motif is unwittingly present in 48% of the most potent antisense oligonucleotides reported in the literature. This finding was prospectively used to predict the sequences of additional antisense oligonucleotides for the rat TNF-alpha primary RNA transcript. Over 50% of antisense constructs (13 of 22) containing the TCCC motif were found to effectively inhibit TNF-alpha synthesis. Marked reductions in mRNA were also observed. This motif was found to be most effective when targeting introns in the primary RNA transcript, suggesting a nuclear localization for the antisense action. Predicting target sites based on the presence of this motif in primary RNA transcripts should be of value in the development on new antisense pharmacotherapy.

Animals↗
PubMed1992

The effect of loop size in antisense and target RNAs on the efficiency of antisense RNA control.

Most natural antisense RNAs display a high degree of secondary structure with stem-loops as their most prominent feature. Mutations affecting the inhibitory activity of these RNAs most often map in or close to loop regions in both the antisense and target RNAs. The primary recognition loops often contain 5-7 unpaired nucleotides. Nucleotide changes in the loops affect the binding rate and, hence, the inhibitory effect on the activity of the target RNA. Here we address the question whether loop sizes affect binding rates between antisense and target RNAs, using the replication control system of plasmid R1 as a model system. By creating a series of loop size mutants we show that loop size alterations have strong effects on the binding rates between the two reactant RNAs in vitro, and that most of the mutations analyzed display corresponding effects on antisense RNA control in vivo. Our data suggest that the three-dimensional structures of antisense and target RNA stem-loops are crucial for determining binding rates. The implications of these results for the design of efficient artificial antisense RNA control systems are discussed.

Bacterial Proteins↗
PubMed2002

Expression of antisense RNA targeted against Streptococcus thermophilus bacteriophages.

Antisense RNA complementary to a putative helicase gene (hel3.1) of a cos-type Streptococcus thermophilus bacteriophage was used to impede the proliferation of a number of cos-type S. thermophilus bacteriophages and one pac-type bacteriophage. The putative helicase gene is a component of the Sfi21-type DNA replication module, which is found in a majority of the S. thermophilus bacteriophages of industrial importance. All bacteriophages that strongly hybridized a 689-bp internal hel3.1 probe were sensitive to the expression of antisense hel3.1 RNA. A 40 to 70% reduction in efficiency of plaquing (EOP) was consistently observed, with a concomitant decrease in plaque size relative to that of the S. thermophilus parental strain. When progeny were released, the burst size was reduced. Growth curves of S. thermophilus NCK1125, in the presence of variable levels of bacteriophage kappa3, showed that antisense hel3.1 conferred protection, even at a multiplicity of infection of approximately 1.0. When the hel3.1 antisense RNA cassette was expressed in cis from the kappa3-derived phage-encoded resistance (PER) plasmid pTRK690::ori3.1, the EOP for bacteriophages sensitive to PER and antisense targeting was reduced to between 10(-7) and 10(-8), beyond the resistance conferred by the PER element alone (less than 10(-6)). These results illustrate the first successful applications of antisense RNA and explosive delivery of antisense RNA to inhibit the proliferation of S. thermophilus bacteriophages.

Amino Acid Sequence↗
PubMed1999

Prediction of antisense oligonucleotide binding affinity to a structured RNA target.

Antisense oligonucleotides, which act through the pairing of complementary bases to an RNA target sequence, are showing great promise in research and clinical applications. However, the selection of effective antisense oligonucleotides has proven more difficult than initially presumed. We developed a prediction algorithm to identify those sequences with the highest predicted binding affinity for their target mRNA based on a thermodynamic cycle that accounts for the energetics of structural alterations in both the target mRNA and the oligonucleotide. The model was used to predict the binding affinity of antisense oligonucleotides complementary to the rabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNFalpha) mRNAs, for which large experimental datasets were available. Of the top ten candidates identified by the algorithm for the RBG mRNA, six were the most strongly binding sequences determined from an experimental assay. The prediction for the TNFalpha mRNA also identified high affinity sequences with approximately 60% accuracy. Computational prediction of antisense efficacy is more cost-efficient and faster than in vitro or in vivo selection and can potentially speed the development of sequences for both research and clinical applications.

Animals↗