Determination of cellular internalization of fluoresceinated oligonucleotides.
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Biological Transport↗
Biomedical subjects
C A Stein
Publications and source records attributed to C A Stein.
Is irrelevant cleavage the price of antisense efficacy?
Antisense oligonucleotides are useful reagents for the suppression of gene expression. Their mechanism of action in eukaryotic cells appears to depend heavily on the activity of RNase H, a ubiquitous enzyme that cleaves the mRNA strand of an RNA-DNA duplex. However, the stringency requirements of RNase H are very low, and as little as a 5-base complementary region of oligomer to target may be sufficient to elicit RNase H activity. This would result in scission of nontargeted mRNAs, or what is known as "irrelevant cleavage." One strategy to reduce RNase H competency that has been employed is modification of the oligonucleotide backbone, replacing phosphodiester linkages with uncharged methylphosphonates, which are not RNase H competent. Another strategy involves replacement of deoxyribonucleic acid with 2'-O-alkylribonucleic acid. A third strategy, eliminating RNase H dependency entirely, requires activation of RNase P. The relative merits of these strategies will be discussed in the context of selective inhibition of gene function.
DNA↗
Cellular delivery of antisense oligonucleotides.
Antisense oligonucleotides can be successfully employed to inhibit specifically gene expression. However, many oligonucleotide classes are polyanions and cannot passively transit the cell membrane. Thus, the use of naked oligonucleotides for antisense purposes poses some rather stringent challenges, and it is not a trivial task to appropriately interpret the data derived from experiments in which they have been used. Multiple methods have been developed to improve intracellular, and in particular, intranuclear oligonucleotide delivery, and in doing so, to maximize the performance of the antisense technologies that are currently available. This review discusses the use of cationic lipids, protein and peptide delivery agents, and several novel chemical and viral methods that have recently been explored as delivery vehicles, focussing not only on their strengths, but also on their limitations.
Animals↗
Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences.
Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such "irrelevant cleavage" would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5' terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2'-O-methyl oligoribonucleotide targeted to the 3' region of the PKC-alpha mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-alpha protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.
3' Untranslated Regions↗
Two problems in antisense biotechnology: in vitro delivery and the design of antisense experiments.
Antisense oligonucleotides are invaluable reagents for the specific downregulation of gene expression. In the absence of a carrier, charged oligonucleotides (e.g., phosphorothioates) can interact with a large number of cell surface proteins, but tend to be internalized into the endosomal/lysosomal compartment. However, they can be successfully delivered to the nuclei of diverse cell types via the use of a wide variety of reagents, including cationic lipids, and cationic polyamines. Over the past several years, a more general understanding of the rules governing the interpretation of data derived from antisense experiments has been reached. These are discussed with emphasis on how to avoid some of the confounding features of this important, emerging technology.
Animals↗
Inhibition of heparanase activity and tumor metastasis by laminarin sulfate and synthetic phosphorothioate oligodeoxynucleotides.
Heparanase activity correlates with the metastatic potential of tumor cells. Moreover, the anti-metastatic effect of non-anti-coagulant species of heparin and certain sulfated polysaccharides was attributed to their heparanase-inhibiting activity. We investigated the effect of a chemically sulfated polysaccharide (laminarin), consisting primarily of beta-1,3 glucan (sodium laminarin), and of synthetic phosphorothioate oligodeoxynucleotides, primarily phosphorothioate homopolymer of cytidine (SdC28), on heparanase activity and tumor metastasis. Investigation of the ability of tumor cells to degrade heparan sulfate in intact extracellular matrix revealed that heparanase activity expressed by B16-BL6 mouse melanoma cells and 13762 MAT rat mammary adenocarcinoma cells was effectively inhibited by LS (50% inhibition at 0.2-1 microgram/ml), but there was no inhibition by sodium laminarin up to a concentration of 50 microgram/ml. Complete inhibition of the melanoma heparanase was obtained in the presence of 0.1 microM SdC28. A single i.p. injection of laminarin sulfate, but not of sodium laminarin, before i.v. inoculation of the melanoma or breast-carcinoma cells inhibited the extent of lung colonization by the tumor cells by 80 to 90%. Similar inhibition was exerted by 0.1 microM SdC28. At the effective concentrations, both compounds had a small effect on proliferation of the tumor cells and on growth of the primary tumors in vivo. These results further emphasize the involvement of heparanase in tumor metastasis and the potential clinical application of diverse heparanase-inhibiting molecules such as sulfated polysaccharides and synthetic polyanionic molecules.
Animals↗
Omega-6 polyunsaturated fatty acid-stimulated cellular internalization of phosphorothioate oligodeoxynucleotides: evidence for protein kinase C-zeta dependency.
The rate of cellular internalization of phosphorothioate oligodeoxynucleotides is determined predominantly by adsorptive plus fluid-phase endocytosis. Internalization of a 5'-fluoresceinated phosphorothioate 15mer homopolymer of thymidine (FSdT15) in K562 cells in medium containing lipid-depleted albumin was reduced consistently versus nondepleted albumin. Treatment of K562 and several other cell lines with omega-6 polyunsaturated fatty acids (omega-6 PUFAs; e.g. arachidonic and linoleic acids) but not saturated fatty acids dramatically increased FSdT15 internalization in a concentration-dependent manner and over a wide albumin concentration range. The rate of efflux of FSdT15 from K562 cells was not affected by the omega-6 PUFA, implying that an increase of cellular fluorescence was due to an increase in the in-rate. These data were consistent with the observation that the binding of FSdT15 to the cell surface was also increased in the presence of omega-6 PUFAs. Omega-6 PUFAs are stimulators of protein kinase C (PKC) activity. Inhibition of PKC activity in K562 cells by Go6976, an inhibitor of the classical PKC isoforms, did not block the linoleic acid-induced stimulation of FSdT15 internalization. On the other hand, treatment of cells with Ro318220, which has considerably less isoform specificity, almost totally blocked the effect of linoleic acid on FSdT15 internalization, implying the involvement of a nonclassical PKC isoform in the process. Finally, since the only PKC isoform expressed in K562 cells that also is activated by omega-PUFAs is PKC-zeta, we obtained NIH 3T3 cells expressing a doxycycline-repressible dominant negative PKC-zeta mutant. Expression of the mutant blocked the stimulation of FSdT15 internalization by linoleic acid. Stimulated internalization also was blocked by wortmannin and LY 294002, which are relatively specific inhibitors of phosphatidylinositol 3-kinase (PI 3-K). Taken together, our data suggest that omega-6 PUFA stimulation of fluoresceinated phosphorothioate oligomers may be PKC-zeta dependent, and perhaps PI-3K dependent as well.
Ceramides↗
Highly loaded nanoparticulate carrier using an hydrophobic antisense oligonucleotide complex.
Antisense oligonucleotides, and particularly those with phosphorothioate backbones, have emerged as potential gene specific therapeutic agents and are currently undergoing evaluation in clinical trials for a variety of diseases. In the area of HIV-1 therapeutics, targeting of oligonucleotides to infected cells, such as macrophages, would be highly desirable. The present study was designed to prepare and characterize oligonucleotide-loaded nanoparticles for this purpose. Due to their hydrophilic characteristics, oligonucleotides are difficult to entrap in polymeric particles. Here, the oligonucleotides were first complexed with cetyltrimethylammonium bromide. The oligonucleotide-loaded nanoparticles were prepared by the emulsification-diffusion method and subsequently purified. In comparison with previous studies, a high oligonucleotide-loading was achieved; 2.5, 5 and 10% oligonucleotide loading were assessed. If the initial oligonucleotide content was 4%, this method produced a final oligonucleotide loading of 1.9% with an entrapment efficiency of 47%. The integrity of the oligonucleotide and of the polymer, in the final freeze-dried product, was retained.
Cetrimonium↗
Uptake of oligonucleotide-loaded nanoparticles in prostatic cancer cells and their intracellular localization.
The development of antisense biotechnology is dependent, in part, on creating improved methods for delivering oligonucleotides to cells. In this study, we investigated a colloidal system (nanoparticles (NP) of poly (D,L) lactic acid) that affects the intracellular delivery of oligonucleotides. We have examined the intracellular compartmentalization in DU145 cells of fluorescein labeled phosphorothioate oligonucleotides, both in the free state and when loaded into NP. Fluorescent oligonucleotides were incubated for 18 h with DU145 cells and the mean intracellular fluorescence was determined by flow cytometry. After the addition of monensin, an increase in signal intensity was observed, indicating that free oligonucleotides were resident in an acidic intracellular environment, whereas oligonucleotides from the NP did not reside in an acidic compartment. Free and NP loaded with oligonucleotides effluxed from DU145 cells from two intracellular compartments. This preliminary report indicates that colloidal carriers such as NP could prove to be useful in affecting intracellular trafficking of oligonucleotides in DU145 and in other cells.
Humans↗
Hybridization prediction gets to first base.
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Nucleic Acid Conformation↗
Keeping the biotechnology of antisense in context.
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Biotechnology↗
Antisense strategies to inhibit restenosis.
Restenosis after percutaneous transluminal coronary angioplasty (PTCA) and coronary stenting remains a major clinical problem. Vascular smooth muscle cell (SMC) proliferation and migration from the arterial wall media into the intima are believed to play a critical role in the pathogenesis of restenosis. Several studies have demonstrated that phosphorothioate (PS) oligodeoxynucleotides targeted against genes involved in SMC proliferation inhibit in vitro SMC proliferation and migration. Moreover, PS oligodeoxynucleotides targeted against the genes c-myb, c-myc, cdc2 kinase, cdk2 kinase, and proliferating cell nuclear antigen (PCNA) when delivered adventitially or intraluminally inhibit in vivo neointimal formation after balloon injury in both the rat carotid and porcine coronary artery models. The inhibitory effects of these PS oligodeoxynucleotides may be the result of their suppression of migration of medial SMC rather than suppression of medial or intimal cell proliferation. Other studies have demonstrated the presence of the potent guanosine or G-quartet aptameric inhibitory effect of the PS oligodeoxynucleotides. Experiments with cytidine homopolymers such as S-dC28, which lack guanosines, reveal the presence of potent non-G-quartet, non-sequence-specific inhibitory effects on in vitro SMC proliferation, migration, and adhesion as well as in vivo neointimal formation after rat carotid artery balloon injury. This is owing to the avid binding of these PS oligodeoxynucleotides to the SMC mitogens and chemoattractants platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF). The extent to which hybridization-dependent antisense, G-quartet aptameric, or non-G-quartet, non-sequence-specific inhibitory effects occurs is the result of PS oligodeoxynucleotide sequence, length, and concentration. The 18-mer guanosine-rich PS oligodeoxynucleotide ZK10 is a more potent in vitro SMC proliferation inhibitor than S-dC28, although both compounds manifest comparable in vivo inhibitory effects on neointimal formation in the rat carotid artery model of balloon injury. PS oligodeoxynucleotides also possess non-sequence-specific immunomodulatory effects, including the induction of interferon-gamma and the unmethylated CpG motif, which exhibits numerous immunomodulatory effects. Novel strategies to inhibit restenosis include the development of E2F transcription decoys that inhibit several cell cycle regulatory genes and diminish neointimal lesion formation. In addition, antisense oligonucleotides targeted against the anti-apoptotic gene bcl-xL, which when transfected into the vessel wall inhibits bcl-xl expression, induce a five-fold increase in apoptotic SMC intimal cells, and effect a marked attenuation of in vivo lesion dimensions, thereby suggesting frank vascular lesion regression.
Animals↗
Porous balloon delivery of S-dC28 does not prevent restenosis in the porcine coronary artery model of balloon injury.
Phosphorothioate (PS) oligodeoxynucleotides (ODN) inhibit vascular smooth muscle cell proliferation through antisense and G-quartet aptameric mechanisms. PS-ODN such as the cytidine homopolymers, have been demonstrated to have non-G-quartet, nonsequence-specific inhibitory effects in a rat carotid balloon injury model of neointimal proliferation. We sought to test the efficacy of S-dC28, a cytidine homopolymer lacking G-quartets, on neointimal proliferation in the porcine coronary artery model of balloon injury. A total of 23 animals (11 controls, 12 treated) were subjected to balloon injury in a coronary artery, followed by infusion of control solution or S-dC28 via porous balloon, the Scimed Dispatch Coronary Infusion Catheter. After a mean interval of 49 days, the animals were killed, and the target coronary segments were examined histologically. S-dC28 did not significantly inhibit neointimal formation. Fluorescein isothiocyanate (FITC)-labeled S-dC28 was present in the intima and media immediately after administration but was present mainly within the adventitia 3 hours after administration. S-dC28, when delivered by a Scimed Dispatch Coronary Infusion Catheter (Maple Grove, MN), did not significantly affect neointimal proliferation after balloon injury in a porcine coronary artery model.
Angioplasty, Balloon, Coronary↗
Paradoxical increase in retinoblastoma protein in colorectal carcinomas may protect cells from apoptosis.
The retinoblastoma (Rb) gene is inactivated in a variety of human cancers, but in colorectal carcinomas there is frequently increased expression of this gene. This is paradoxical in view of the known role of Rb as a tumor suppressor gene. In the present study, we compared the levels of expression of the Rb protein (pRb) in normal human colorectal mucosa, adenomatous polyps, and carcinomas by immunohistochemistry. In vitro studies were also done to examine the phenotypic effects of an antisense oligodeoxynucleotide (AS-Rb) targeted to Rb mRNA in the HCT116 colon carcinoma cell line that expresses a relatively high level of pRb. The incidence of pRb-positive cells was increased during multistage colorectal carcinogenesis. In vitro treatment of HCT116 cells with AS-Rb decreased the level of pRb by about 70% and also decreased the levels of the cyclin D1 protein and cyclin D1-associated kinase activity. AS-Rb inhibited growth of HCT116 cells and induced apoptosis. Reporter assays indicated about a 17-fold increase in E2F activity. These findings suggest that the increased expression of pRb in colorectal carcinoma cells may provide a homeostatic mechanism that protects them from growth inhibition and apoptosis, perhaps by counterbalancing potentially toxic effects of excessive E2F activity.
Apoptosis↗
Mechanisms of action of taxanes in prostate cancer.
Taxanes appear to have significant clinical activity in hormone-refractory prostate cancer and are entering increasing numbers of clinical trials. In general, they appear to initiate the apoptotic process by binding to beta-tubulin and promoting its polymerization. However, it is possible, at least in prostate cancer cell lines, that the ultimate decision for or against apoptosis may be modulated by a number of factors, including levels of Bcl-family members, especially the antiapoptotic proteins bcl-2 and bcl-xL. Differences in the cellular pharmacology of docetaxel (Taxotere; Rhône-Poulenc Rorer, Collegeville, PA) and paclitaxel have been identified. Docetaxel has a somewhat higher affinity for microtubules than paclitaxel (approximately twofold), and in some circumstances may be a more potent inducer of bcl-2 phosphorylation. However, it is not clear whether these docetaxel-induced events are directly related to its antiprostate cancer effects. In murine macrophages, paclitaxel, but not docetaxel, induces several proinflammatory genes, but it is not known whether this occurs in prostate cancer cells. The mechanisms of cytotoxicity of taxanes in prostate cancer are undoubtedly complex and will require considerable additional study to fully understand.
Antineoplastic Agents, Hormonal↗
Cationic porphyrins: novel delivery vehicles for antisense oligodeoxynucleotides.
Cationic porphyrins form stable complexes with oligodeoxynucleotides. To evaluate delivery, we used a 20mer phosphorothioate oligomer (Isis 3521) targeted to the 3'-untranslated region of the PKC-alpha mRNA, and complexed it with porphyrin. The expression of PKC-alpha protein and mRNA in T24 bladder carcinoma cells was reduced by approximately 80 +/- 10% at a concentration of oligomer of 3 microM, and 9 microM porphyrin. The expression of PKC-beta1, -delta and -straightepsilon isoforms was unaffected by this treatment, but elimination of PKC-zeta protein and mRNA were observed. However, treatment with the porphyrin complex of Isis 3522, an oligomer which is directed at the 5' coding region of the PKC-alpha mRNA, was equally effective as Isis 3521 with respect to PKC-alpha, but did not affect PKC-zeta protein or mRNA levels. Since Isis 3521 has an 11-base region of complementarity with the PKC-zeta mRNA, wheras Isis 3522 has only a 4-base region, the effect of Isis 3521 on PKC-zeta protein and mRNA expression may be due to irrelevant cleavage. Depending upon the desired application, this new strategy may offer several advantages over other methods of antisense oligodeoxynucleotide delivery including efficiency, stability, solubility, relatively low toxicity and serum compatibility. Porphyrins may thus be a potentially useful delivery vehicle for antisense therapeutics and/or target validation.
Energy Transfer↗
The extracellular domain of CD4 receptor possesses a protein kinase activity.
The CD4 receptor of T-helper cells is an essential participant in immune response formation and HIV infection. We report here that the extracellular domains of CD4 receptor can catalyze the phosphotransferase (kinase) reaction. Incubation of rsCD4 in solution with [gamma-32P]ATP results in the Ca2+-dependent autophosphorylation of the protein presumably at a His residue because the reaction is prevented by the diethylpyrocarbonate treatment. The rsCD4 phosphorylates milk casein or human plasma proteins as a Ser/Thr protein kinase.
Adenosine Triphosphate↗
How to design an antisense oligodeoxynucleotide experiment: a consensus approach.
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Drug Design↗