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Results for “antisense targeting”

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PubMed1999

Antisense oligodeoxynucleotides targeted to MAG mRNA profoundly alter BP and PLP mRNA expression in differentiating oligodendrocytes: a caution.

The applicability of antisense technology to suppress the expression of myelin associated glycoprotein (MAG) in cultured oligodendrocytes was evaluated. Differentiating oligodendrocyte precursor cells obtained by the shake-off method were exposed to nine unmodified antisense oligodeoxynucleotides (ODNs) targeted to the first seven exons of MAG mRNA. After four days, steady-state levels of MAG, proteolipid protein (PLP) and basic protein (BP) mRNAs were determined by Northern blot analysis. Only ODN annealing to 599-618 nt of the MAG mRNA (the junction of exon 5 and 6) resulted in a significant, 75% decrease in the MAG mRNA level. Unexpectedly, six other anti-MAG ODNs which had no significant effect on the MAG message, greatly increased the level of BP mRNA. The highest upregulation of approximately 12 fold was observed with ODN annealing to 139-168 nt (junction of exon 3 and 4). On the other hand, the 997-1016 ODN decreased the levels of BP and PLP messages by 70-80%. The 599-618 ODN also decreased the PLP mRNA by 85%. The results demonstrate that antisense ODNs targeted to one gene may profoundly alter the expression of other genes, and hence, complicate functional analysis of the targeted protein.

Animals↗

PubMed2005

Synergistic antitumor effect of combined use of adenoviral-mediated p53 gene transfer and antisense oligodeoxynucleotide targeting clusterin gene in an androgen-independent human prostate cancer model.

Our recent studies showed that antisense oligodeoxynucleotide targeting antiapoptotic gene, clusterin, enhanced apoptosis induced by conventional therapeutic modalities using several prostate cancer models. In this study, to establish a more effective therapeutic strategy against prostate cancer, we investigated the effect of combined treatment with antisense clusterin oligodeoxynucleotide and adenoviral-mediated p53 gene transfer (Ad5CMV-p53) in an androgen-independent human prostate PC3 tumor model. Treatment of PC3 cells with 500 nmol/L antisense clusterin oligodeoxynucleotide decreased clusterin mRNA by >80% compared with that with 500 nmol/L mismatch control oligodeoxynucleotide. Clusterin mRNA expression in PC3 cells was highly up-regulated by Ad5CMV-p53 treatment; however, antisense clusterin oligodeoxynucleotide treatment further suppressed clusterin expression in PC3 cells after Ad5CMV-p53 treatment. Antisense clusterin oligodeoxynucleotide treatment significantly enhanced the sensitivity of Ad5CMV-p53 in a dose-dependent manner, reducing the IC50 of Ad5CMV-p53 by 75%. Apoptotic cell death was detected after combined treatment but not after treatment with either agent alone. In vivo administration of antisense clusterin oligodeoxynucleotide and Ad5CMV-p53 resulted in a significant inhibition of s.c. PC3 tumor growth as well as lymph node metastases from orthotopic PC3 tumors compared with administration of either agent alone. Furthermore, combined treatment with antisense clusterin oligodeoxynucleotide, Ad5CMV-p53, and mitoxantrone completely eradicated s.c. PC3 tumors and lymph node metastases from orthotopic PC3 tumors in 60% and 100% of mice, respectively. These findings suggest that combined treatment with antisense clusterin oligodeoxynucleotide and Ad5CMV-p53 could be a novel strategy to inhibit progression of hormone-refractory prostate cancer and that further addition of chemotherapeutic agents may help to enhance the efficacy of this combined regimen.

Adenoviridae↗

PubMed2003

An antisense plasmid targeting survivin expression induces apoptosis and sensitizes hepatocarcinoma cells to chemotherapy.

To explore the change of sensitivity to chemotherapy of antisense RNA targeting survivin on hepatocarcinoma carcinoma cells in vitro. Survivin mRNA structure region was amplified by RT-PCR and inserted inversely into eukaryotic expression vector pcDNA3. The antisense expression plasmid pcDNA3/survivin was transfected into HepG2 with lipofectAMINE 2000 (LF2000), with low concentration of 5-fluorouracil (5-Fu) added. Survivin protein was detected by Western-blot, the growth activity was measured by MTT, and apoptosis was detected by Flow Cytometry 12 h, 24 h, 48 h after transfection. The activity of caspase-3 was found by quantitative assay 48 h after transfection. The construction of antisense RNA vector pcDNA3/survivin was verified by restricted endonuclease digestion and nucleotide sequencing. Compared with normal group, 5-Fu and antisense survivin group, the cells growth inhibition, apoptosis index, and caspase-3 activity were increased in antisense survivin transfected + 5-Fu group. The threshold of apoptosis was decreased after survivin was silenced, and the sensitivity to chemotherapy was increased. These findings suggest the existence of a potential new target for gene therapy.

Antigens, Neoplasm↗

PubMed1997

Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A.

Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIalpha regulatory subunit results in cancer cell growth inhibition in vitro and in vivo. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G2 phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained in vivo in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.

Animals↗

PubMed1993

Computer-aided search for effective antisense RNA target sequences of the human immunodeficiency virus type 1.

For the biological and therapeutic application of antisense nucleic acids, there is a need to identify effective local target regions of given cellular target mRNAs or viral single-stranded nucleic acids. One critical parameter for the effectiveness of antisense nucleic acids could be the potential of intramolecular folding of a given sequence element of the target strand and the antisense strand, respectively. The folding potential of such subsequences was calculated by using an established secondary structure prediction algorithm. For the genomic RNA and the complementary RNA strand of the human immunodeficiency virus type 1 (HIV-1), an energy profile was calculated that monitors the local folding potential of each sequence position surrounded by a window of given length ranging from 50 to 400 nucleotides. The resulting energy profile was compared to the effectiveness of HIV-1-directed antisense RNAs. It was found that significant minima of the local folding potential (high delta G values) correlated with antisense RNA target regions involved in strong inhibition of HIV-1 replication that had been measured independently in two earlier studies by using different experimental approaches. Conversely, antisense RNAs directed against local subregions with a high folding potential (low delta G values) showed weak or no antiviral effect in human cells. The results indicate that analyses of the local folding potential of a given target RNA can support the selection of effective target sequences for antisense RNA.

Antiviral Agents↗

PubMed2006

A phase I safety and dose escalation trial of docetaxel combined with GEM231, a second generation antisense oligonucleotide targeting protein kinase A R1alpha in patients with advanced solid cancers.

PURPOSE: GEM231 is a second-generation antisense oligonucleotide targeting the mRNA of the R1alpha regulatory subunit of cAMP dependent protein kinase A. Preclinical studies have demonstrated synergistic antitumor activity when GEM231 is combined with docetaxel. This trial assesses the safety of this combination. EXPERIMENTAL DESIGN: Docetaxel was administered once every three weeks (one-cycle) at doses between 50-75 mg/m2. GEM231 was administered twice weekly at 220 mg/m2 for 3 (schedule-A), or 2 (schedule-B) weeks. RESULTS: Twenty patients with chemotherapy-refractory advanced cancer received a total of 39 cycles of therapy. Six patients in schedule-A received docetaxel 50 mg/m2, and 14 patients in schedule-B received docetaxel 50-75 mg/m2. In schedule-A, 2 of 6 patients developed cycle-1 dose limiting toxicity (DLT)-grade-3 fatigue or grade-3 serum transaminase elevation. In schedule-B, 1 of 4 patients developed cycle-1 DLT at the highest dose of docetaxel tested (75 mg/m2)--grade-3 febrile neutropenia. Subsequent dose escalations were not pursued since the overall incidence of grade-3 toxicities (including those that occurred after cycle 1) was 75%, and this dose was close to the single agent MTD of docetaxel. Grade-3 toxicities included fatigue (2 patients), transaminase elevation (4 patients), and altered mentation (1 patient). The mean post-infusion aPTT was significantly higher than the pre-infusion value [14.8 seconds; p<0.001]; however, there were no hemorrhagic episodes. CONCLUSIONS: The recommended dose for further development of the combination of docetaxel and GEM231 is 75 mg/m2 and 220 mg/m2, respectively. It is important to administer GEM231 twice weekly for 2 consecutive weeks followed by a one-week break.

Adult↗

PubMed2004

[Optimal effects of liposome on antisense oligodeoxynucleotides uptake by mouse breast cancer TM40D cells and the effects of antisense oligodeoxynucleotides targeting c-erbB-2 on cell proliferation and apoptosis].

OBJECTIVE: To study the optimal effects of liposome on antisense oligodeoxynucleotides targeting c-erbB-2 uptake by mouse breast cancer TM40D cells and effects of AS-ODNs on cell proliferation and apoptosis. METHODS: Flow cytometric analysis and fluorescence microscope were used to study the transfection ratio of AS-ODNs in mouse breast cancer TM40D cells with/without liposome in 1, 2, 4, 6 hour and the distribution of AS-ODNs in cells. the effects of ODNs on cell proliferation and activation of apoptosis were examined by MTT assay and flow cytometry. RESULTS: Liposome could promote AS-ODNs uptake by mouse breast cancer TM40D cells, flow cytometric analysis revealed that the time of the optimal effects was 4 hours and the percentage of FAM-positive cells reached 72.23%; the ratio maintained at a relatively high level at 6 h. Also liposome facilitated the entrance of AS-ODNs into nucleus. AS-ODNs restrained the proliferation of mouse breast cancer TM40D cells, and restraint rate was 50.24%. Meanwhile AS-ODNs promoted cell apoptosis. Flow cytometry analysis revealed that antisense ODNs increased cell apoptosis by 38.50%, compared with cultured cells group 9.13% and liposome group 9.29%. CONCLUSIONS: Liposome could facilitate AS-ODNs to enter cells and their nucleus. AS-ODNs of C-erbB-2 restrained the proliferation of mouse breast cancer TM40D cells and promoted apoptosis.

Animals↗

PubMed2001

Antinociceptive effects after intrathecal administration of phosphodiester-, 2'-O-allyl-, and C-5-propyne-modified antisense oligodeoxynucleotides targeting the NMDAR1 subunit in mouse.

In the present study, we have compared the antinociceptive effect of three different types of antisense oligodeoxynucleotides targeting the N-methyl-D-aspartate (NMDA) R1-subunit in mice. The probes were administrated intrathecally three times during a period of 5 days (1, 5 or 25 microg/injection), followed by evaluation using the formalin test. The antinociceptive effect was correlated to in vitro receptor binding in spinal cord sections. The tissue distribution was studied after a single injection of fluorescein-conjugated probes. The phosphodiester probe showed superficial tissue penetration after 30 min and disappeared within 2 h. The probe did, however, significantly reduce both receptor binding in laminae I and II (by 36-44% compared to saline) as well as pain behavior (32% compared to saline), without apparent side effects. The mismatched probe was ineffective at 25 microg, while some reductions in receptor binding and pain behavior were seen after 5 microg. The C-5-propyne-modified phosphorothioate probe showed pronounced tissue penetration and cellular uptake as soon as 30 min after injection which was still detectable after 24 h. Immediately after injection of the highest dose, long-lasting hind-limb paralysis was observed. Receptor binding was reduced but not in a dose-related manner. Pain behavior was significantly reduced by 40% following 25 microg of antisense probe but not after lower doses or 25 microg of mismatched probe. The 2'-O-allyl-modified probe did not significantly reduce receptor binding or pain behavior. Thus, only the phosphodiester probe showed a significant correlation between reduction in pain behavior and receptor binding. These findings demonstrate that antisense technology is associated with specificity problems, but still could provide a valuable tool to study the role of different target proteins in the drug discovery process.

2-Amino-5-phosphonovalerate↗

PubMed2004

Antisense RNA targeting of primase interferes with bacteriophage replication in Streptococcus thermophilus.

The putative primase gene and other genes associated with the Sfi21-prototype genome replication module are highly conserved in Streptococcus thermophilus bacteriophages. Expression of antisense RNAs complementary to the putative primase gene (pri3.1) from S. thermophilus phage kappa 3 provided significant protection from kappa 3 and two other Sfi21-type phages. Expression of pri3.10-AS, an antisense RNA that covered the entire primase gene, reduced the efficiency of plaquing (EOP) of kappa 3 to 3 x 10(-3) and reduced its burst size by 20%. Mutant phages capable of overcoming antisense inhibition were not recovered. Thirteen primase-specific antisense cassettes of different lengths (478 to 1,512 bp) were systematically designed to target various regions of the gene. Each cassette conferred some effect, reducing the EOP to between 0.8 and 3 x 10(-3). The largest antisense RNAs (1.5 kb) were generally found to confer the greatest reductions in EOP, but shorter (0.5 kb) antisense RNAs were also effective, especially when directed to the 5' region of the gene. The impacts of primase-targeted antisense RNAs on phage development were examined. The expression of pri3.10-AS resulted in reductions in target RNA abundance and the number of phage genomes synthesized. Targeting a key genome replication function with antisense RNA provided effective phage protection in S. thermophilus.

Base Sequence↗

PubMed2004

Targeting of antisense PNA oligomers to human galanin receptor type 1 mRNA.

In this work, we have targeted positions 18-38 of the human galanin receptor type 1 (GalR1) mRNA coding sequence with different peptide nucleic acid (PNA) oligomers. This region has previously been shown to be a good antisense region and therefore we aimed to identify the subregions and/or thermodynamic parameters determining the antisense efficacy. Nine different PNA oligomers were conjugated to a cell-penetrating peptide, transportan, to enhance their cellular uptake. Concentration-dependent down-regulation of GalR1 protein expression in human melanoma cell line Bowes was measured by radioligand binding assay. No reduction of GalR1 mRNA level was observed upon PNA treatment, thus, the effect was concluded to be translational arrest. Judging from the EC50 values, antisense PNA oligomers targeting regions 24-38 (EC50=70 nM) or 27-38 (EC50=80 nM) were the most potent suppressors of protein expression. No parameter predicted by M-fold algorithm was found to correlate with the measured antisense activities. Presence of some subregions was found not to increase antisense efficiency of PNA. Presence of a short unpaired triplet between nucleotides 33 and 35 in the target region was, on the other hand, found to be the most critical for efficient GalR1 down-regulation. Thus, the results are of high impact in designing antisense oligomers. Specific results of this study demonstrate 20-fold more efficient antisense down-regulation of GalR1 as achieved before.

Cell Line, Tumor↗

PubMed1998

Anti-proliferative effects of unmodified antisense oligodeoxynucleotides targeted against c-raf mRNA: use of poly (lysine/serine) copolymers or cationic lipopolyamines.

1. It is now known that nuclease-resistant phosphorothioate antisense oligodeoxynucleotides (ODN) have some actions that are unrelated to antisense mechanisms. In the present study we assessed the anti-proliferative effects of phosphorothioate (PS) and phosphodiester (PO; unmodified) antisense ODN targeted against c-raf mRNA on pancreatic cancer cells in vitro, using poly (lysine/serine) copolymers conjugated with polyethylene glycol (PLSP) or cationic lipopolyamines (Transfectam) as carriers. 2. The anti-proliferative effect of the PO antisense ODN was significantly (P < 0.05) greater than that of the PS ODN, either complexed with PLSP (2 mumol/L ODN) or the Transfectam (0.5 mumol/L ODN). However, the effect of the PS or PO antisense ODN was not dependent on the antisense sequence. The c-raf mRNA levels, assessed by reverse transcription-polymerase chain reaction, were obviously reduced by both PO and PS antisense ODN compared with mismatched ODN when complexed with the Transfectam (1 mumol/L ODN). 3. Although the anti-proliferative effects were mainly unrelated to antisense mechanisms, unmodified antisense ODN complexed with some carriers could be used as anti-tumour agents considering that synthetic carriers can be modified to improve functions, such as delivery.

Cations↗

PubMed1999

Antisense oligodeoxynucleotides targeting internal exon sequences efficiently regulate TNF-alpha expression.

ABSTRACT Exon sequences upstream of splice sites play a critical role in mRNA processing, which is dependent on spliceosome interactions with these sites. Using antisense oligodeoxynucleotides (ODN), we targeted these and other sequences of the proinflammatory tumor necrosis factor-alpha (TNF-alpha) gene because it is multiply spliced and has been difficult to regulate with ODN in the past. ODN targeting exon sequences upstream of the donor splice sites of internal exons 2 (ORF4) and 3 (ORF6) significantly reduced TNF-alpha levels in stimulated U937 cells by 62%+/-7% and 51%+/-9%, respectively, in a dose-dependent manner but did not affect interleukin-6 (IL-6) levels. In contrast, ODN targeting the exon sequences downstream of the acceptor splice sites of exons 1, 2, and 3 failed to reduce TNF-alpha levels significantly under the same conditions. End-phosphorothioated ORF4 (ORF4-PE) significantly reduced TNF-alpha mRNA levels by greater than 80% (p < 0.001) and protein levels by 60% (p < 0.001) in U937 cells. ORF4-PE reduced newly synthesized TNF-alpha protein levels by >80% in lipopolysaccharide (LPS)-stimulated human macrophages, by greater than 60% in phorbol myristate acetate/phyto-hemagglutinin (PMA/PHA)-stimulated human peripheral blood mononuclear cells (PBMC), and by approximately 50% in LPS-stimulated murine monocytes. These results suggest that exon sequences flanking donor splice sites are highly susceptible target domains for antisense inhibition of TNF-alpha gene expression.

Animals↗

PubMed2000

Non-specific antiviral activity of antisense molecules targeted to the E1 region of human papillomavirus.

Antisense phosphorothioate oligonucleotides (ODN1 0x5 OMe) directed against the E1 start region of human papillomavirus 11 (HPV11) can inhibit papillomavirus induced growth of implanted human foreskin in a mouse xenograft model. Administration of a mismatch control oligonucleotide (ODN9 0x5 OMe), in which guanine was replaced with adenine in the same model, had no effect on papilloma induced growth. However, the apparent antiviral activity of ODN1 0x5 OMe was also shown in a lethal mouse cytomegalovirus (CMV) model, in which the oligonucleotides are not expected to have antisense activity. To understand the mechanisms of action of these oligonucleotides, a mismatch oligonucleotide (ODN61 0x5 OMe) was prepared which retained the CpG motifs of ODN1 0x5 OMe. This was tested in the mouse xenograft model and shown to have moderate inhibitory activity. As a definitive experiment, a comparison was made between the efficacy of the active oligonucleotide ODN1 0x5 OMe against two papilloma viruses HPV11 and HPV40. Both these viruses cause benign genital warts, but differ by four bases in their E1 sequence that was the target for ODN1 0x5 OMe. Papillomavirus induced growth in the mouse xenograft model was inhibited by ODN1 0x5 OMe in both cases, suggesting that oligonucleotide molecules have a non-specific antiviral activity that is not directly related to their antisense sequence.

Animals↗

PubMed2006

Antisense oligonucleotides targeting midkine induced apoptosis and increased chemosensitivity in hepatocellular carcinoma cells.

AIM: Overexpression of midkine (MK) has been observed in many malignancies. This aim of this study is to screen for suitable antisense oligonucleotides (ASODN) targeting MK in hepatocellular carcinoma (HCC) cells and evaluate its antitumor activity. METHODS: Ten ASODN targeting MK were designed and synthesized. After transfection with ASODN, cell proliferation was analyzed with MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. In addition, MK mRNA, protein levels, as well as apoptosis and caspase-3 activity were also examined in HepG2 cells. Cell proliferation was then analyzed after treatment with both ASODN and chemotherapeutic drugs. RESULTS: In this experiment, the ASODN5 among the 10 ASODN showed higher inhibitory activity against proliferation of hepatocellular carcinoma cells in a dose-dependent manner. In HepG2 cells, ASODN5 could significantly reduce the MK mRNA level and protein content. After transfection with ASODN5 for 48 h, accompanied with a decline of survivin and Bcl-2 protein content, a remarkable increase of apoptosis and caspase-3 activity was observed in HepG2 cells. Furthermore, ASODN5 transfer can significantly increase chemosensitivity in HepG2 cells. CONCLUSION: Antisense oligonucleotides targeting MK shows therapeutic effects on HCC; ASODN5 has the possibility to be developed as an effective antitumor agent.

Antimetabolites, Antineoplastic↗

PubMed1999

Antitumor activity and pharmacokinetics of a mixed-backbone antisense oligonucleotide targeted to the RIalpha subunit of protein kinase A after oral administration.

Overexpression of the RIalpha subunit of cAMP-dependent protein kinase (PKA) has been demonstrated in various human cancers. PKA has been suggested as a potential target for cancer therapy. The goal of the present study was to evaluate an anti-PKA antisense oligonucleotide (mixed-backbone oligonucleotide) as a therapeutic approach to human cancer treatment. The identified oligonucleotide inhibited the growth of cell lines of human colon cancer (LS174T, DLD-1), leukemia (HL-60), breast cancer (MCF-7, MDA-MB-468), and lung cancer (A549) in a time-, concentration-, and sequence-dependent manner. In a dose-dependent manner, the oligonucleotide displayed in vivo antitumor activity in severe combined immunodeficient and nude mice bearing xenografts of human cancers of the colon (LS174T), breast (MDA-MB-468), and lung (A549). The routes of drug administration were intraperitoneal and oral. Synergistic effects were found when the antisense oligonucleotide was used in combination with the cancer chemotherapeutic agent cisplatin. The pharmacokinetics of the oligonucleotide after oral administration of (35)S-labeled oligonucleotide into tumor-bearing mice indicated an accumulation and retention of the oligonucleotide in tumor tissue. This study further provides a basis for clinical studies of the antisense oligonucleotide targeted to the RIalpha subunit of PKA (GEM 231) as a cancer therapeutic agent used alone or in combination with conventional chemotherapy.

Administration, Oral↗

PubMed1997

Abnormal patterning of the aortic arch arteries does not evoke cardiac malformations.

Ablation of the cardiac neural crest results in abnormal development of the aortic arch arteries leading to altered patterning of the great arteries. The cardiac outflow tract is also affected after neural crest ablation because normally a subset of neural crest cells migrates from the pharyngeal region to form the outflow septum. Using neural crest ablation, it has not been possible to separate the occurrence of aortic arch maldevelopment from cardiac outflow tract dysmorphogenesis. In order to determine whether normal aortic arch artery development is a prerequisite for normal outflow tract development, we have used a combination of antisense treatment with backtransplantation of cardiac neural folds to produce abnormal patterning of the aortic arch arteries. Paralogous groups of Hox messages with their anterior expression domains in pharyngeal arches 3, 4 and 6 were targeted. Antisense targeted to paralogous group 3 Hox message caused aortic arch 3 located within the pharyngeal arch to regress in a manner similar to aortic arch 2, while antisense targeted to paralogous group 5 Hox message caused the appearance of an additional pharyngeal arch containing a novel and completely independent aortic arch artery. Antisense treatment targeting paralogous group 4 Hox message led to no detectable cardiovascular phenotype in the first 6 days of development. While regression of arch artery 3 was associated with abnormal branching patterns of the aorta and pulmonary trunk, this did not involve abnormal separation of the aorta and pulmonary trunks, the semilunar valves or the subvalvular region of the outflow tract. Because none of these changes in pharyngeal or aortic arch artery development was accompanied by abnormal development of the cardiac outflow tract, it appears that normal patterning of the aortic arch arteries is not a prerequisite for normal heart development. Using reverse transcription polymerase chain reaction (RT-PCR) we were unable to detect changes in any of the Hox messages except group 4, thus, using this particular experimental strategy, we are unable to demonstrate or refute that expression of hox genes by cardiac neural crest cells controls aortic arch patterning. Development of the cardiac outflow tract was normal in each instance. This suggests that abnormal aortic arch patterning does not necessarily lead to cardiac malformations.

Animals↗

PubMed2003

Administration of dendritic cells transduced with antisense oligodeoxyribonucleotides targeting CD80 or CD86 prolongs allograft survival.

BACKGROUND: The expression of costimulatory molecules on antigen-presenting cells is crucial in determining T-cell immune responses. We examined the effects of transduction with high-affinity antisense oligodeoxyribonucleotides (ODNs) designed to target the mRNA of CD80 or CD86 on the phenotype and function of dendritic cells (DCs). MATERIALS AND METHODS: DCs were propagated from C57BL/10 (B10; H2b) bone marrow cells in granulocyte macrophage-colony stimulating factor and interleukin (IL)-4, and transduced with anti-CD80 or anti-CD86 antisense ODNs (base-mismatched ODNs as controls). The effect of antisense ODN on phenotype was examined by flow cytometry. The allostimulatory function of DCs was assessed by mixed leukocyte reaction and cytotoxic activity in vitro, and the influence on allograft survival was assessed in vivo. RESULTS: ODNs were effectively incorporated by DCs, which were enhanced by the presence of lipofectamine. Antisense ODNs targeting CD80 or CD86 mRNA specifically suppressed the expression of CD80 or CD86 in DCs and inhibited their capacity to elicit the proliferative responses, donor-specific cytotoxic T-lymphocyte activity in C3H (H2k) spleen T cells. This was associated with decreased IL-2, but elevated IL-4 production, and an increase in T-cell apoptosis. In contrast with the administration of control DCs into C3H recipients that exacerbated rejection of B10 cardiac allografts, injection of DCs transduced with anti-CD80 or CD86 antisense ODN significantly prolonged the survival of heart allografts. CONCLUSION: Transduction with antisense ODN targeting CD80 or CD86mRNA is a feasible and effective approach to modify DCs that renders them tolerogenic by inducing T-cell hyporesponsiveness and apoptosis. This may lead to the development of new therapeutic strategies in transplantation.

Animals↗

PubMed2005

Antisense oligodeoxynucleotides targeting the serine/threonine kinase Pim-2 inhibited proliferation of DU-145 cells.

AIM: To investigate the effect of antisense oligodeoxynucleotides (ASODN) targeting Pim-2 on cell proliferation of DU-145 cells. METHODS: Three ASODN targeting Pim-2 were designed and synthesized. After transfection with ASODN, cell proliferation was analyzed using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. In addition, Pim-2 mRNA, protein levels, and cell cycles were examined. RESULTS: The ASODN designed and synthesized by our laboratory significantly reduced Pim-2 mRNA level and protein content in DU-145 cells. After transfection with ASODN for 48 h, a marked reduction in cell viability was observed in DU-145 cells in a dose-dependent manner. No remarkable apoptosis occurred in cells treated with ASODN compared with control cells. However, it should be noted that G1 phase arrest was clearly observed in ASODN-treated cells. CONCLUSION: ASODN targeting Pim-2 resulted in a marked reduction in DU-145 cell proliferation, and induction of G1 phase cell cycle arrest is one of the important mechanisms for ASODN to reduce cell growth. Moreover, antisense inhibition of Pim-2 expression provides a new promising therapy target for prostate cancer.

Apoptosis↗