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Results for “Oligodeoxyribonucleotides, Antisense”

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At least 19 recordsLinked to original sources
PubMed2000

[Bcl-2 antisense oligodeoxyribonucleotide increases apoptosis of lung carcinoma cells induced by cisplatin].

OBJECTIVE: To study the effect of antisense oligodeoxyribonucleotide (ODN) to apoptotic suppress gene bcl-2 on apoptosis of lung carcinoma cells induced by cisplatin. METHODS: The lung carcinoma cells expressing bcl-2 were chosen to participate in this experiment. Cultured cells were divided into 7 groups: ODN, nonsense, ODN + cisplatin, nonsense + cisplatin, cisplatin, lipofectin and control. Bcl-2 antisense or nonsense mixed with lipofectin was added into above corresponding cultured cells. After cultured for 6 hours, cisplatin was added into corresponding groups. The cells were cultured again for 16 hours. And then, the cells were smeared on slides. Apoptotic cells were labeled with TdT-mediated dUTP nick end labeling (TUNEL) method on cell smears. Apoptotic index (AI) was counted to show the percentage of apoptotic cancer cells. The immunocytochemistry was used to detect the expression of bcl-2 in carcinoma cells. RESULTS: The bcl-2 expression of cancer cell in ODN group was significantly decreased compared to the control and nonsense groups. The AI of ODN + cisplatin group was 16.4 +/- 1.7, cisplatin group 4.1 +/- 0.8, antisense group 5.9 +/- 0.2, nonsense group 3.3 +/- 0.7, nonsense + cisplatin 7.6 +/- 1.1, lipofectin 5.1 +/- 0.9, control group 3.6 +/- 0.6. The AI of antisense + cisplatin group was significantly higher than that of other groups. CONCLUSION: Antisense oligodeoxyribonucleotide to bcl-2 can inhibit significantly the expression of bcl-2 of lung cancer cells and increase apoptosis of cancer cells induced by cisplatin.

Antineoplastic Agents↗
PubMed2005

Inhibitory effect of human telomerase antisense oligodeoxyribonucleotides on the growth of gastric cancer cell lines in variant tumor pathological subtype.

AIM: To investigate the inhibitory effect of specialized human telomerase antisense oligodeoxyribonucleotides on the growth of well (MKN-28), moderately (SGC-7901) and poorly (MKN-45) differentiated gastric cancer cell lines under specific conditions and its inhibition mechanism, and to observe the correlation between the growth inhibition ratio and the tumor pathologic subtype of gastric cancer cells. METHODS: Telomerase activity in three gastric cancer cell lines of variant tumor pathologic subtype was determined by modified TRAP assay before and after the specialized human telomerase antisense oligodeoxyribonucleotides were dealt with under specific conditions. Effect of antisense oligomer under specific conditions of the growth and viability of gastric cancer cell lines was explored by using trypan blue dye exclusion assay, and cell apoptosis was detected by cell morphology observation, flow cytometry and TUNEL assay. RESULTS: Telomerase activity was detected in well, moderately and poorly differentiated gastric cancer cell lines (the quantification expression of telomerase activity was 43.7TPG, 56.5TPG, 76.7TPG, respectively). Telomerase activity was controlled to 30.2TPG, 36.3TPG and 35.2TPG for MKN-28, SGC-7901 and MKN-45 cell lines respectively after treatment with human telomerase antisense oligomers at the concentration of 5 mumol/L, and was entirely inhibited at 10 mumol/L, against the template region of telomerase RNA component, whereas no inhibition effect was detected in missense oligomers (P<0.05). After treatment with antisense oligomers at different concentrations under specific conditions for 96 h, significant growth inhibition effects were found in MKN-45 and SGC-7901 gastric cancer cell lines (the inhibition ratio was 40.89% and 71.28%), but not in MKN-28 cell lines (15.86%). The ratio of inactive SGC-7901 cells increased according to the prolongation of treatment from 48 to 96 h. Missense oligomers could not lead to the same effect (P<0.05). Apoptosis of SGC-7901 and MKN-45 cells was detected not only by morphology and TUNEL assay but also by flow cytometry. The apoptotic rate reached 33.56% for SGC-7901 cells and 44.75% for MKN-45 cells. CONCLUSION: The viability and proliferation of gastric cancer cells can be inhibited by antisense telomerase oligomers. The growth inhibition of gastric cancer cells is correlated with concentration, time and sequence specialty of antisense oligomers. The inhibition mechanism of antisense human telomerase oligomers depends not only on the sequence specialty but also on the biological characteristics of gastric cancer cell lines.

Apoptosis↗
PubMed2001

Specific inhibition of estrogen receptor alpha function by antisense oligodeoxyribonucleotides.

We have tested the effect of a range of antisense oligodeoxyribonucleotides (ODN) directed against the human estrogen receptor alpha (ERalpha) on ERalpha protein expression and function. Antisense ERalpha ODN transfected into the ERalpha-positive human breast carcinoma cell line MCF7-K2 showed variable responses dependent on the oligo used. The most active antisense ODN (oligo 7) decreased the levels of ERa protein by 61% as measured by Western blot analysis. Exogenous 17beta-estradiol (17beta-E2), but not 17alpha-E2, augmented this effect, with a threshold effect at 10(-8) M 17beta-E2. The inhibitory effect of antisense ERa oligo 7 was confirmed by measurement of functional ERalpha protein. 3H-17beta-E2 binding to MCF7 cell extracts was inhibited to approximately 40% of control values in the presence of oligo 7. Antisense-transfected MCF7-K2 cell cultures produced a further 30% binding reduction in the presence of exogenous 17beta-E2. An inhibitory effect on 17beta-E2-dependent cell function was confirmed by the demonstration that ERalpha oligo 7-transfected MCF7-K2 cells failed to exhibit 17beta-E2-stimulated cell proliferation. Exogenous 17beta-E2 enhanced the inhibitory effect of the antisense ODN by increasing ODN transfection efficiency but without ERalpha catabolism via the proteosomal pathway, suggesting an effect of 17beta-E2 on the plasma membrane and the existence of different ERalpha degradation pathways in the MCF7-K2 cell subclone. As 17beta-E2 had no effect on ERalpha protein degradation, we conclude that the observed reduction of ERalpha protein levels is due solely to the presence of the antisense ERalpha ODN. Antisense ERalpha ODN molecules, therefore, may form the basis of effective therapies against ERalpha-dependent malignancies.

Adenocarcinoma↗
PubMed2003

Sensitization and apoptosis augmentation of K562/ADM cells by anti-multidrug resistance gene peptide nucleic acid and antisense oligodeoxyribonucleotide.

AIM: To investigate the reversal effect and apoptosis enhancement of peptide nucleic acid (PNA) and antisense oligodeoxyribonucleotide (ASODN) targeted to multidrug resistance gene (mdr1) on human multidrug resistant leukemia K562/ADM cells. METHODS: A 15-mer PNA and the same sequence of ASODN, complementary to the 5' end of the AUG initiator codon-containing region of mdr1 messenger RNA (MDR1-PNA, MDR1-ASODN), were designed and synthesized. Proliferation and sensitivity to adriamycin of K562/ADM cells treated with MDR1-PNA- and MDR1-ASODN were analyzed with a MTT colorimetric assay. Apoptotic morphologies, P-glycoprotein (P-gp) expression, intracellular adriamycin accumulation, and cell cycle were measured. RESULTS: MDR1-PNA 1 to 10 micromol/L and MDR1-ASODN 2 to 20 micromol/L alone had no inhibitory effects on the proliferation of K562/ADM cells, but significantly inhibited the growth of K562/ADM cells cultured in adriamycin-containing medium. After treatment with MDR1-PNA and MDR1-ASODN, intracellular adriamycin accumulation in K562/ADM cells increased greatly and P-gp synthesis was strikingly reduced. The resistance to adriamycin of the drug-resistant cells was partly reversed and the cells were induced to apoptosis by adriamycin. The reversal efficacy of MDR1-PNA was 3.1-fold higher than that of the same sequence of MDR-ASODN, but neither MDR1-PNA nor MDR1-ASODN could completely block the mdr1/P-gp expression. CONCLUSION: Sequence-special PNA targeted to mdr1 gene more effectively than the same sequence of MDR1-ASODN inhibited the expression of P-glycoprotein to overcome the drug-resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Neutralization of tumor necrosis factor alpha (TNF alpha) action on cell proliferation in rat blastocysts by antisense oligodeoxyribonucleotides directed against TNF alpha p60 receptor.

Antisense oligodeoxyribonucleotide inhibition of gene expression was used to test whether the p60 form of the tumor necrosis factor alpha (TNF alpha) receptor (Rp60) is responsible for mediating the negative effect of TNF alpha on the development of rat blastocysts in vitro. The antisense oligonucleotide was designed to overlap the translation initiation codon of the TNF alpha Rp60 mRNA. Preliminary experiments showed that concentrations of oligonucleotides above 10 microM in the culture medium were embryotoxic over 24 h. When used at nontoxic concentrations (8 microM), antisense oligonucleotides specifically decreased the abundance of intact TNF alpha Rp60 transcripts by 80% within 3 h of exposure. In contrast to results with control embryos, mRNA for the second form of TNF alpha receptor, TNF alpha Rp80, was detected in blastocysts exposed to antisense oligonucleotides to TNF alpha Rp60. Antisense oligonucleotides to TNF alpha Rp60 blocked the 25-30% decrease in cell proliferation induced by 50 ng/ml TNF alpha added to a standard culture medium and by 5 ng/ml TNF alpha added to a medium that had been conditioned by rat uterine cells. Sense oligonucleotides had no such protective effect. Because uterine cells from diabetic rats secrete higher levels of TNF alpha than those from control rats, antisense oligonucleotides were also tested in a medium that had been conditioned by diabetic uterine cells (cell-secreted TNF alpha concentration was 50 pg/ml in this medium, and no exogenous TNF alpha was added). Addition of antisense oligonucleotides to TNF alpha Rp60 improved the quality of this medium with respect to cell proliferation but failed to correct the high frequency of dead cells observed in the blastocysts.

Animals↗
PubMed2000

Human hepatoma cell telomerase activity inhibition and cell cycle modulation by its RNA component antisense oligodeoxyribonucleotides.

AIM: To investigate the effects of human telomerase RNA component antisense oligodeoxyribonucleotides on telomerase activity of human hepatoma cells and their effects on cell cycle distribution. METHODS: Modified telomeric repeat amplification protocol was used to detect telomerase activity. Cell cycle was analyzed by flow cytometer. RESULTS: Telomerase activity was detected in all of four human hepatoma cell lines but absent in normal liver cells. Antisense oligomers to human telomerase RNA component (hTR) inhibited telomerase activity of BEL-7404 human hepatoma cells markedly in vitro. After in vitro treatment with antisense oligomers for 96 h, cell cycle of BEL-7404 human hepatoma cells was mainly arrested at G2/M phase. CONCLUSION: Antisense oligomers to hTR inhibited telomerase activity of BEL-7404 human hepatoma cells in vitro and resulted in cell cycle arrest at G2/M phase.

Carcinoma, Hepatocellular↗
PubMed2000

[Plasmodium falciparum: antimalarial activities of antisense oligodeoxyribonucleotides targeted to histidine-rich protein genes in vitro].

OBJECTIVE: To study the inhibition of growth of Plasmodium falciparum cultured in vitro with antisense oligodeoxyribonucleotide (AS ODN) against histidine-rich proteins (HRP). METHODS: The ODNs against HRP II and HRP III were synthesized and used to study the antimalarial activities in vitro. Plasmodium falciparum (FCC1/HN strain, China) were exposed to AS DONs for 48 h, and the growth inhibition was determined by microscopic examination. RESULTS: At 1 mumol/L, all ODNs inhibited parasite growth and development in a target-independent manner. However, when the ODN concentrations were between 0.01 and 0.5 mumol/L, the AS ODN significantly inhibited the growth and development of P. falciparum compared with ODN controls (P < 0.01). Inhibition by the sense strand ODN did not differ significantly from the control group (P > 0.05). CONCLUSION: Blockade of the expression of HRP II and HRP III AS ODN could inhibit P. falciparum cultured in vitro.

Animals↗
PubMed1999

A theoretical approach to select effective antisense oligodeoxyribonucleotides at high statistical probability.

Up to now, out of approximately 20 antisense oligodeoxyribonucleotides (as ODN) selected and tested against a given target gene, only one species shows substantial suppression of target gene expression. In part, this seems to be related to the general assumption that the structures of local target sequences or antisense nucleic acids are unfavorable for efficient annealing. Experimental approaches to find effective as ODN are extremely expensive when including a large number of antisense species and when considering their moderate success. Here, we make use of a systematic alignment of computer-predicted secondary structures of local sequence stretches of the target RNA and of semi-empirical rules to identify favorable local target sequences and, hence, to design more effective as ODN. The intercellular adhesion molecule 1 (ICAM-1) gene was chosen as a target because it had been shown earlier to be sensitive to antisense-mediated gene suppression. By applying the protocol described here, 10 ICAM-1-directed as ODN species were found that showed substantially improved inhibition of target gene expression in the endothelial cell line ECV304 when compared with the most effective published as ODN. Further, 17 out of 34 antisense species (50%) selected on the theoretical basis described here showed significant (>50%) inhibition of ICAM-1 expression in mammalian cells.

Cell Line, Transformed↗
PubMed1993

Predictions of effect for intracellular antisense oligodeoxyribonucleotides from a kinetic model.

We have analyzed the implications of a simple two-compartment mathematical model (Hargrove and Schmidt, 1989) to anticipate the limits of antisense oligodeoxyribonucleotide action within single cells. The steady-state equations are derived for four special cases representing the following mechanisms: (i) ribosome blockage, (ii) mRNA cleavage by RNase H, (iii) concurrent ribosome exclusion and RNase H action, and (iv) decreased delivery of mature mRNA to the cytoplasm due to transcriptional blockage, interference with nucleocytoplasmic transport, or splicing. Dose-response relationships have been derived for these mechanisms under ideal conditions. Our results indicate that frequently translated mRNAs producing stable proteins are the most attractive antisense targets because these protein levels are sensitive to the changes in the mRNA levels that can be effected using antisense oligodeoxyribonucleotides. The nonsteady-state solutions show that both mRNA and protein half-life can determine the kinetics of antisense oligonucleotide action. A rapid onset of effect will be observed when the mRNA is rapidly degraded and slowly translated and when the translated protein is rapidly degraded. When the protein is slowly degraded, the kinetics of effect are limited by protein half-life. When the translational rate constant is large compared to the absolute difference between the mRNA and protein degradation rate constants, the kinetics of antisense action are determined by both degradation rate constants but are limited by the slower of the two degradative processes. We also show that the steady-state and nonsteady-state solutions may be used to design experiments that discriminate among mechanisms of antisense action.

Algorithms↗
PubMed1999

Clinical use of streptolysin-O to facilitate antisense oligodeoxyribonucleotide delivery for purging autografts in chronic myeloid leukaemia.

Antisense oligodeoxyribonucleotides (ODN) targeted against the breakpoint in BCR-ABL mRNA will specifically decrease BCR-ABL mRNA, provided cells are first permeabilised with streptolysin-O (SL-O). We used 18-mer chimeric methylphosphonodiester: phosphodiester linked (4-9-4) ODN complementary to 9 bases either side of the BCR-ABL junction to purge harvests ex vivo in three CML patients who remained completely Ph positive after multiple chemotherapy courses. After CD34+ cell selection and SL-O permeabilisation, harvests were purged with 20 microM ODN. After purging, all individual CFU-GM colonies grown from the two b3a2 breakpoint cases remained positive for BCR-ABL mRNA. In contrast, all 24 colonies grown from the b2a2 breakpoint case were BCR-ABL mRNA negative. Patients were conditioned with busulphan 16 mg/kg. The initial post-transplant course was uneventful, although the time to return to 0.5 x 10(9)/l neutrophils was slow at 25-51 days. Both chronic phase patients remain in haematological remission at +724 and +610 days, although each has cytogenetic evidence of relapse. The b2a2 accelerated phase patient died of myeloid blast transformation at day +91. The present SL-O-facilitated ODN purging strategy appears to be without significant toxicity, and offers considerable improvements in ODN delivery to the cytosol.

Antigens, CD34↗
PubMed1997

Antisense oligodeoxyribonucleotides suppress hematologic cell growth through stepwise release of deoxyribonucleotides.

Antisense oligodeoxyribonucleotides (ODNs) are now being extensively investigated in an attempt to achieve cell growth suppression through specific targeting of genes related to cell proliferation, despite increasing evidence of non-antisense cytotoxic effects. In the context of anti-BCR/ABL antisense strategies in chronic myeloid leukemia, we have reexamined the antiproliferative effect of phosphodiester and phosphorothioate ODNs on the leukemic cell line BV173 and on CD34+ bone marrow cells in liquid culture. The 3' sequences of the ODNs determine their effect. At concentrations of 10 micromol/L (for phosphorothioate ODNs) or 25 micromol/L (for phosphodiester ODNs), all the tested ODNs exert an antiproliferative activity, except those that contain a cytosine residue at either their two most terminal 3' positions. We show that this antiproliferative effect is due to the toxicity of the d-NMPs (5' monophosphate deoxyribonucleosides), the enzymatic hydrolysis products of the ODNs in culture medium. The toxicity of the d-NMPs on hematologic cells depends on their nature (d-CMP [2'deoxycytidine 5'-monophosphate] is not cytotoxic), on their concentration (d-GMP [2'-deoxyguanosine 5'-monophosphate], TMP [thymidine 5'-monophosphate], and d-AMP [2'-deoxyadenosine 5'-monophosphate] are cytotoxic at concentrations between 5 and 10 micromol/L), and on the coincident presence of other d-NMPs in the culture medium (d-CMP neutralizes the toxicity of d-AMP, d-GMP, or TMP). The antiproliferative activity of ODNs is thus restricted to conditions where the 3' hydrolysis process by exonucleases generates significant amounts of d-NMPs with a low proportion of d-CMP. Our results reveal a novel example of a nonantisense effect of ODNs, which should be taken into account when performing any experiment using assumed antisense ODNs.

Cell Division↗
PubMed2006

Treatment of Streptococcus mutans with antisense oligodeoxyribonucleotides to gtfB mRNA inhibits GtfB expression and function.

We examined the effects of phosphorothioate-modified antisense oligodeoxyribonucleotides (PS-ODN) targeted to mRNA transcribed from gtfB, which encodes synthesis of water-insoluble glucans in Streptococcus mutans. Treatment of S. mutans with 10 muM antisense PS-ODNs inhibited gtfB mRNA transcription, GtfB expression and water-insoluble glucan synthesis. The architecture of biofilms formed by antisense PS-ODNs-treated S. mutans showed reduced biomass, more dispersed distribution with enlarged interspaces and fewer layers of attached cells. PS-ODN treatment had no effect on the growth of S. mutans. Our results indicated that it might be feasible to use antisense PS-ODN as a novel agent in caries prevention.

Adjuvants, Pharmaceutic↗
PubMed1988

The inhibition of neurite outgrowth in PC12 cells by tubulin antisense oligodeoxyribonucleotides.

A model system was designed to determine the effect of antisense oligodeoxyribonucleotides (oligos) on the expression of endogenous tubulin genes, the major component of microtubules, and to study the involvement of individual tubulin isoforms in specific functions, by employing antisense oligos which can block the expression of specific transcripts, both in vitro and in vivo. In reticulocyte cell-free system, specific inhibition of mRNA translation was observed with oligos complementary to the 5' alpha-tubulin-coding region. In vivo inhibition of neurite outgrowth was observed in nerve growth factor-induced PC12 cells. Specific inhibition was observed in cells treated with antisense oligos corresponding to the 5' alpha-tubulin-coding region as well as with antisense oligos corresponding to the 3' alpha-noncoding regions of two different alpha-tubulin isoforms. The results show that both tubulin isoforms are involved in neurite outgrowth and further demonstrate the ability of oligos to block expression of endogenous genes and thus follow the involvement of those gene products in cellular differentiation.

Adrenal Gland Neoplasms↗
PubMed1992

Sequence-dependent cleavage of DNA by alkylation with antisense oligodeoxyribonucleotides containing a 2-(N-iodoacetylaminoethyl)thio-adenine.

Antisense oligodeoxyribonucleotides (15mers), containing a 2-(N-iodoacetylaminoethyl)thio-adenine, were synthesized and tested for their ability to cleave complementary DNAs (21mers). Cleavage of the target DNAs was done by alkylation followed by treatment with piperidine, and the positions of the alkylated sites were estimated by identification of the cleaved products. By using several combinations of the modified strands and their target DNAs, it was determined that alkylation occurred at adenine or guanine, depending on the torsion angle of the modified nucleoside.

Adenine↗
PubMed2005

[Study on the modulation of estrogen receptor isoforms alpha in endometrial carcinoma cells using antisense oligodeoxyribonucleotides].

OBJECTIVE: To verify the roles of estrogen receptor alpha in the tumogenesis of endometrial cancer in relationship with estrogen and tamoxifen, and to explore an efficient way to modulate the expression of estrogen receptor (ER) isoform alpha to built up a model of endometrial cancer cells expressing predominant one isoform of ERs for ultimately probing therapeutical and preventive antiestrogen for endometrial cancer. METHODS: A series of oligodeoxyribonucleotides against regions of the human ERalpha was synthesized and tested in human endometrial cancer cell line (HEC-1B cells) that expressed functional ERalpha. The expressions of ER isoforms were detected by Western blot using specific antibody. The change of HEC-1B cell proliferation in response to 17beta-estradiol(E2) and tamoxifen(TAM) under the impact of antisense oligodeoxyribonucleotides (AS-ODN) was studied. RESULTS: (1) Transfection with AS-ODN directed against the human ERalpha significantly inhibited ERalpha protein expression. (2) The proliferation of endometrial cancer cell line HEC-1B could be increased by both E(2) and TAM. The cells lost the ability to proliferate in response to E(2) after being transfected with ERalpha-AS-ODN at hours 24, 48 and 72. After being transfected with ERalpha-AS-ODN, HEC-1B cells lost the ability to proliferate in response to TAM at hour 48. CONCLUSION: (1) AS-ODNs directed against the human ERalpha can inhibit the expression of ER alpha effectively. The method can be used to built up a model of endometrial cancer cells expressing predominant one isoform of ERs. (2) ERalpha may be the primary receptor in the proliferation of HEC-1B cells in response to 17beta-estradiol and tamoxifen.

Cell Proliferation↗