Harnessing the power of antisense technology for combination chemotherapy.
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Animals↗
Biomedical subjects
R Narayanan
Publications and source records attributed to R Narayanan.
Cell cycle-dependent modulation of telomerase activity in tumor cells.
Telomerase is a ribonucleoprotein complex that is thought to add telomeric repeats onto the ends of chromosomes during the replicative phase of the cell cycle. We tested this hypothesis by arresting human tumor cell lines at different stages of the cell cycle. Induction of quiescence by serum deprivation did not affect telomerase activity. Cells arrested at the G1/S phase of the cell cycle showed similar levels of telomerase to asynchronous cultures; progression through the S phase was associated with increased telomerase activity. The highest level of telomerase activity was detected in S-phase cells. In contrast, cells arrested at G2/M phase of the cell cycle were almost devoid of telomerase activity. Diverse cell cycle blockers, including transforming growth factor beta1 and cytotoxic agents, also caused inhibition of telomerase activity. These results establish a direct link between telomerase activity and progression through the cell cycle.
Antineoplastic Agents↗
Immunocytochemical localization of the endogenous neuroexcitotoxin quinolinate in human peripheral blood monocytes/macrophages and the effect of human T-cell lymphotropic virus type I infection.
Quinolinate (Quin), a metabolite in the kynurenine pathway of tryptophan degradation and a neurotoxin that appears to act through the N-methyl-D-aspartate receptor system, was localized in cultured human peripheral blood monocytes/macrophages (PBMOs) by using a recently developed immunocytochemical method. Quin immunoreactivity (Quin-IR) was increased in gamma interferon (IFN-gamma)-stimulated monocytes/macrophages (MOs). In addition, the precursors, tryptophan and kynurenine, significantly increased Quin-IR. Infection of MOs by human T-cell lymphotropic virus type I (HTLV-I) in vitro substantially increased both the number of Quin-IR cells and the intensity of Quin-IR. At the peak of the Quin-IR response, about 40% of the cells were Quin-IR positive. In contrast, only about 2-5% of the cells were positive for HTLV-I, as detected by both immunofluorescence for the HTLV-I antigens and PCR techniques for the HTLV-I Tax gene. These results suggest that HTLV-I-induced Quin production in MOs occurs by an indirect mechanism, perhaps via cytokines produced by the infection but not directly by the virus infection per se. The significance of these findings to the neuropathology of HTLV-I infection is discussed.
Cells, Cultured↗
Multiple mechanisms may contribute to the cellular anti-adhesive effects of phosphorothioate oligodeoxynucleotides.
Phosphorothioate oligodeoxynucleotides complementary to the p65 (Rel A) subunit of the NF-kappaB nuclear transcriptional regulatory factor have been suggested to be sequence specific blockers of cellular adhesion. We studied the effects of Rel A antisense, Rel A sense and other phosphorothioate oligodeoxynucleotides on cellular adhesion and found that blockade of adhesion was predominately non-sequence specific. Phosphorothioate oligodeoxynucleotides bind to the extracellular matrix (ECM) of NIH 3T3 cells, and to the ECM elements laminin and fibronectin. By use of a gel mobility shift assay, the association of the A subunit of laminin with a probe 12mer phosphodiester oligodeoxynucleotide could be demonstrated. This interaction was described by a single-site binding equation (K d = 14 microM). Human Rel A antisense and sense oligodeoxynucleotides, and two synthetic persulfated heparin analogs were excellent competitors of the binding of the probe oligodeoxynucleotide to laminin. Taken together, these data indicate that oligodeoxynucleotide binding occurred at or near the heparin-binding site. Competition for 5' 32p- SdT18 (an 18mer phosphorothioate homopolymer of thymidine) binding to fibronectin with the discrete heparin analogs, as well as with SdC28, was also observed. Phosphorothioate oligodeoxynucleotides (Rel A antisense >> Rel A sense) inhibited the binding of laminin to bovine brain sulfatide, but not to its cell surface receptors on MCF-7 cells. By flow cytometric analysis we have also shown, in contrast to what was observed with laminin, that phosphorothioates a non-specifically block the specific binding of fluoresceinated fibronectin to its cell surface receptors on phorbol-12,13-myristate acetate treated Jurkat cells. Blockade of specific binding occurred in the oligodeoxynucleotide treated cells in the presence or absence of oligomer in the media.
3T3 Cells↗
On the quality control of antisense oligonucleotides.
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Humans↗
Telomerase as a potential molecular target to study G-quartet phosphorothioates.
Inhibition of gene expression by phosphorothioate oligomers is complex and involves specific and nonspecific mechanisms. Oligomers that contain a G-quartet elicit distinct effects in vitro and in vivo that are dependent on the context of the G-quartet's occurrence within a sequence. The enzyme telomerase, a ribonucleoprotein, has a stretch of C residues in the RNA template, which are used to add terminal dG-rich telomeric repeats to the ends of chromosomes. Some but not all phosphorothioates containing a G-quartet, depending on the context of occurrence, inhibited telomerase activity in vitro. Non-G-quartet phosphorothioates did not inhibit this activity. Activities of control enzymes, such as reverse transcriptase or taq polymerase, were not affected by the G-quartet oligomers. Neither phosphodiester nor chimeric oligomers of a G-quartet-containing oligomer were as potent inhibition of telomerase activity as phosphorothioate oligomers. These results may provide a molecular target to study the effects of G-quartet-containing oligomers.
Animals↗
Antisense therapy.
The sequence specificity of the antisense technique makes it an attractive basis for novel molecular therapeutics. Inhibition of gene expression by antisense in cell culture models has provided a strong rationale for identification and validation of disease targets. Analogues modified from normal phosphodiester oligodeoxynucleotides have entered clinical trials of diseases including AIDS, cancer, and inflammation. It is becoming increasingly apparent that these drugs act by means of a complex mechanism of action, and some of their effects can be sequence independent. Nevertheless, these oligodeoxynucleotides offer considerable promise as novel molecular drugs. Harnessing the therapeutic potential of this powerful technique depends on elucidation of the complex mechanism of action so that effective and meaningful therapeutic modalities can be realized.
Acquired Immunodeficiency Syndrome↗
Increased quinolinate immunoreactivity in the peripheral blood monocytes/macrophages from SIV-infected monkeys.
Quinolinate (QUIN), a metabolite in the kynurenine pathway of tryptophan degradation and a neurotoxin that is thought to act through the NMDA receptor system, was localized in cultured peripheral blood monocytes/macrophages from SIV-infected monkeys using a recently developed immunohistochemical method. Significant increases in QUIN immunoreactive (IR) cells were detected in all five SIV-infected monkeys examined. Multinucleated giant cells, a hallmark of lentiviral infection, were visible in selected samples. Treatment with the QUIN precursors, tryptophan and kynurenine, increased the number of QUIN-IR cells in both the control and SIV-infected preparations, perhaps by a mass action mechanism. We hypothesize that in SIV-infected monkeys, infiltrating monocytes/macrophages contribute to the high level of brain QUIN and associated neuropathology.
Animals↗
Stromal cells regulate bcl-2 and bax expression in pro-B cells.
B lymphocyte production in the bone marrow depends on a cascade of regulatory cells and cytokines unique to the hematopoietic microenvironment. Fibroblastic stromal cells appear to be particularly important in regulating the earliest events in this lineage; however, it is still not clear whether the same or different sets of signals regulate maintenance of cell viability, proliferation, and differentiation of B lineage cells. In this study, we addressed the role of bone marrow stromal cells in survival and expansion of normal murine pro-B cells. Stromal cells were required for long-term proliferation of pro-B cell clone C1.92, and, in the presence of stromal cell line S10, pro-B cells expressed the proto-oncogene bcl-2. Removal of C1.92 cells from Stromal cell-derived signaling in support of pro-B cell viability. Due to its previously described role in regulating cell survival, we investigated whether stromal cells regulate bcl-2 expression in pro-B cells. When removed from stromal cell cultures, pro-B cells rapidly lost bcl-2 mRNA expression coincident with initiation of apoptosis. However, interruption of bcl-2 expression with antisense oligonucleotides in the presence of stroma and interleukin-7 (IL-7) did not result in immediate cell death. Oligonucleotide-treated cells arrested in G(1) phase of the cell cycle 24 hours before the initiation of apoptosis. In contrast, removal of pro-B cells from stromal cell support resulted in rapid increase in BAX expression, correlating directly with initiation of apoptosis. These results suggest that bcl-2 may, in part, regulate cell survival by interrupting the cascade of intracellular events that regulate cell cycle progression in lymphopoietic cells. Initiation of apoptosis in these cells appears to be more closely correlated with intracellular levels of BAX expression.
Animals↗
Differentiation of immortal cells inhibits telomerase activity.
Telomerase, a ribonucleic acid-protein complex, adds hexameric repeats of 5'-TTAGGG-3' to the ends of mammalian chromosomal DNA (telomeres) to compensate for the progressive loss that occurs with successive rounds of DNA replication. Although somatic cells do not express telomerase, germ cells and immortalized cells, including neoplastic cells, express this activity. To determine whether the phenotypic differentiation of immortalized cells is linked to the regulation of telomerase activity, terminal differentiation was induced in leukemic cell lines by diverse agents. A pronounced downregulation of telomerase activity was produced as a consequence of the differentiated status. The differentiation-inducing agents did not directly inhibit telomerase activity, suggesting that the inhibition of telomerase activity is in response to induction of differentiation. The loss of telomerase activity was not due to the production of an inhibitor, since extracts from differentiated cells did not cause inhibition of telomerase activity. By using additional cell lineages including epithelial and embryonal stem cells, down-regulation of telomerase activity was found to be a general response to the induction of differentiation. These findings provide the first direct link between telomerase activity and terminal differentiation and may provide a model to study regulation of telomerase activity.
Animals↗
Sequence context of antisense RelA/NF-kappa B phosphorothioates determines specificity.
The use of antisense oligomers to achieve inhibition of gene expression is complicated by frequent non-specific effects, and even the control oligomers often exhibit sequence-specific effects. We have recently shown that in diverse tumor-derived cell lines, a 24mer phosphorothioate oligomer antisense to the relA subunit of NF-kappa B transcription factor causes a block of cellular adhesion, inhibition of nuclear NF-kappa B and Sp1 DNA-binding activity and inhibition of tumor cell growth in vitro and in vivo. In this study we use the same model to attempt to define the limits of antisense specificity. We demonstrate that single base pair substitution can virtually abolish the antisense activity. The relative position of mismatches within the antisense sequence is critical to the loss of activity. Our results further indicate that antisense specificity is determined not only by the content of the sequence but also by its occurrence with reference to the surrounding sequences.
Animals↗
Transcriptional inhibition of the inducible nitric oxide synthase gene by competitive binding of NF-kappa B/Rel proteins.
The activity of the inducible nitric oxide synthase enzyme (iNOS) is tightly controlled, partly at the transcriptional level. We find NF-kappa B/Rel activation (p50-p50 and p50-p65) in RAW 264.7 macrophages after lipopolysaccharide treatment and binding to both NF-kappa B sites in the mouse iNOS promoter. To delineate the importance of NF-kappa B/Rel in iNOS gene transcription, we used an unusually direct approach to try to improve on the antioxidant-treatment or reporter techniques, namely the depletion of NF-kappa B/Rel activity through the use of a phosphorothioate-modified oligonucleotide containing three copies of the NF-kappa B consensus sequence. The reduction in NF-kappa B/Rel activity (particularly that binding to the downstream of the two sites) was associated with a 50% reduction in NO output and a reduction in the quantity of the iNOS protein expressed. These results point to the probability that physiologically relevant NF-kappa B/Rel activators or repressors other than lipopolysaccharide might crucially affect the macrophage NO response.
Amino Acid Oxidoreductases↗
The therapeutic potential of antisense oligonucleotides.
Specific inhibition of gene expression by antisense agents provides the basis for rational drug discovery based on molecular targets. Due to the specificity of Watson-Crick base-pair hybridization, antisense oligodeoxynucleotides have been used extensively in attempts to inhibit gene expression in both in vitro and in vivo models. Analogues modified from normal phosphodiester oligodeoxynucleotides have entered clinical trials against diseases including AIDS and cancer. Although the precise mechanism of action of these drugs has not been clarified, these oligodeoxynucleotides offer considerable promise as novel molecular therapeutics. We review the recent attempts to harness the therapeutic potential of these oligodeoxynucleotides and appraise the near-term prospects for antisense technology.
Acquired Immunodeficiency Syndrome↗
Thiol agents and Bcl-2 identify an alphavirus-induced apoptotic pathway that requires activation of the transcription factor NF-kappa B.
Oxidative stress has been proposed as a common mediator of apoptotic death. To investigate further the role of oxidants in this process we have studied the effects of antioxidants on Sindbis virus (SV)-induced apoptosis in two cell lines, AT-3 (a prostate carcinoma line) and N18 (a neuroblastoma line). The thiol antioxidant, N-acetylcysteine (NAC), at concentrations above 30 mM, completely abrogates SV-induced apoptosis in AT-3 and N18 cells. The effects of NAC cannot be attributed to inhibition of viral entry or viral replication, changes in extracellular osmolarity or to increases in cellular glutathione levels, nor can they be mimicked by chelators of trace metals, inhibitors of lipid peroxidation or peroxide scavengers. In contrast, other thiol agents including pyrrolidine dithiocarbamate (PDTC, 75 microM) are protective. Because NAC and PDTC are among the most effective inhibitors of the transcription factor NF-kappa B, we examined SV's ability to activate NF-kappa B before the onset of morphologic or biochemical evidence of apoptosis. Within hours of infection, SV induced a robust increase in nuclear NF-kappa B activity in AT-3 and N18 cells; this activation was suppressible by NAC and PDTC. Over-expression of bcl-2 in AT-3 cells, which has been shown to inhibit SV-induced apoptosis, also inhibits SV-induced NF-kappa B activation. To determine if NF-kappa B activation is necessary for SV-induced apoptosis in these cells, we used double stranded oligonucleotides with consensus NF-kappa B sequences as transcription factor decoys (TFDs) to inhibit NF-kappa B binding to native DNA sites. Wild-type, but not mutant, TFDs inhibit SV-induced apoptosis in AT-3 cells. In contrast, TFD inhibition of NF-kappa B nuclear activity in N18 cells did not prevent SV-induced apoptosis. Taken together, these observations define a cell type-specific, transcription factor signaling pathway necessary for SV-induced apoptosis. Understanding the precise mechanism by which Bcl-2 and thiol agents inhibit SV-induced nuclear NF-kappa B activity in AT-3 cells may provide insights into the pluripotent antiapoptotic actions of these agents.
Acetylcysteine↗
Sequence-independent induction of Sp1 transcription factor activity by phosphorothioate oligodeoxynucleotides.
Modified analogues of antisense oligodeoxynucleotides (ODNs), particularly phosphorothioates ([S]ODNs), have been extensively used to inhibit gene expression. The potential sequence specificity of antisense oligomers makes them attractive as molecular drugs for human diseases. The use of antisense [S]ODNs to inhibit gene expression has been complicated by frequent nonspecific effects. In this study we show in diverse cell types that [S]ODNs, independent of their base sequence, mediated the induction of an Sp1 nuclear transcription factor. The [S]ODN-mediated Sp1 induction was rapid and was associated with elevated levels of Sp1 protein. This induction was dependent on NF-kappa B activity, since inhibition of NF-kappa B activity abolished the [S]ODN-induced Sp1 activity. [S]ODN-induced Sp1 activity was seen in mouse spleen cells following in vivo administration. Sp1 activity induced by [S]ODNs required the tyrosine kinase pathway and did not have transactivating potential. These results may help to explain some of the non-specific effects often seen with [S]ODNs.
Animals↗
What is happening to caesarean section rates?
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Cesarean Section↗
In vivo toxicological effects of rel A antisense phosphorothioates in CD-1 mice.
To characterize the in vivo toxicity of phosphorothioate antisense oligonucleotides against rel A (p65 subunit of NF-kappa B transcription factor), forty-eight 6-week-old CD-1 mice were split into 4 groups (6/sex/group) receiving vehicle (phosphate-buffered saline) or doses of 50, 100, and 150 mg/kg of rel A antisense oligonucleotides intraperitoneally 3 times weekly for 2 weeks. Clinical signs of toxicity included weakness, and decreased motor activity and food consumption with body weight loss. Mortality occurred in 7 of 12 mice in the 150-mg/kg group and in 2 of 12 mice in the 100-mg/kg group, most of which died within the first 2 to 4 days of treatment. The remaining mice were necropsied on day 15. The major hematological finding was severe dose-dependent thrombocytopenia. The liver enzyme levels were mildly elevated in the serum of mid- and high-dose animals. At necropsy, increased spleen and liver weights were observed in treated mice, some of which also had mild pleural and/or peritoneal effusions. Histopathological examination revealed the likely cause of death to be acute renal failure due to renal cortical or tubular necrosis. Treatment-related changes were also found in the liver, spleen, bone marrow, and several other organs. In summary, the kidney, liver, and bone marrow (megakaryocytic lineage) were identified as the major target organs for toxicity with rel A antisense therapy.
Animals↗
Antisense oligodeoxynucleotides.
Oligodeoxynucleotides have been proposed as both in vitro and in vivo inhibitors of gene expression because of the specificity of Watson-Crick base pair hybridization. Phosphodiester oligodeoxynucleotides (normal DNA) cannot be used as drugs because they are nuclease sensitive. Significant efforts have been made to study phosphorothioate oligodeoxynucleotides, which have a sulfur atom substituted for one of the phosphate oxygen atoms at a nonbridging position. These oligodeoxynucleotides are nuclease resistant, and over the past year they have entered clinical trials. They have also been extensively examined in vitro and have been targeted to the bcr-abl and bcl2 messenger RNAs among others. Methods to maximize the intracellular oligodeoxynucleotide concentration have also been devised. However, significant problems remain, including the significant nonsequence specificity of phosphodiester oligodeoxynucleotides as well as questions of oligodeoxynucleotide uptake into and compartmentalization within cells. An improvement of our understanding of these phenomena is critical to the elaboration of this technology into a clinical therapeutic modality.
Animals↗