Pub Date : 2002-04-01DOI: 10.1089/108729002760070849
M. Manoharan
This review summarizes the effect of conjugating small molecules and large biomacromolecules to antisense oligonucleotides to improve their therapeutic potential. In many cases, favorable changes in pharmacokinetic and pharmacodynamic properties were observed. Opportunities exist to change the terminating mechanism of antisense action or to enhance the RNase H mode of action via conjugate formation.
{"title":"Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action.","authors":"M. Manoharan","doi":"10.1089/108729002760070849","DOIUrl":"https://doi.org/10.1089/108729002760070849","url":null,"abstract":"This review summarizes the effect of conjugating small molecules and large biomacromolecules to antisense oligonucleotides to improve their therapeutic potential. In many cases, favorable changes in pharmacokinetic and pharmacodynamic properties were observed. Opportunities exist to change the terminating mechanism of antisense action or to enhance the RNase H mode of action via conjugate formation.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"28 1","pages":"103-28"},"PeriodicalIF":0.0,"publicationDate":"2002-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87046444","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-02-01DOI: 10.1089/108729002753670210
Elena Puerta-Fernández, A. Barroso-deljesús, A. Berzal-Herranz
Efficient ribozyme-mediated gene silencing requires the effective binding of a ribozyme to its specific target sequence. Stable stem-loop domains are key elements for efficiency of natural antisense RNAs. This work tests the possibility of using such naturally existing structural motifs for anchoring hairpin ribozymes when targeting long RNAs. Assays were performed with four catalytic antisense RNAs, based on the hairpin ribozyme (HP), that carried a stable stem-loop motif at their 3' end. Extensions consisted of one of the following motifs: the stem-loop II of the natural antisense RNA-CopA, its natural target in CopT, the TAR-RNA motif, or its complementary sequence alphaTAR. Interestingly, the presence of any of these antisense motifs resulted in an enhancement of catalytic performance against the ribozyme's 14-nucleotide-long target RNA (Swt). A series of artificial, long RNA substrates containing the Swt sequence and the natural TAR-RNA stem-loop were constructed and challenged with a catalytic antisense RNA carrying the TAR-complementary stem-loop. This cleaves each of these substrates significantly more efficiently than HP. The deletion of the TAR domain in the substrate, or its substitution by its complementary counterpart alphaTAR, abolishes the positive effect. These results suggest that the enhancement is owed to the interaction of both complementary stem-loop domains. Moreover, they demonstrate that the TAR domain can be used as an anchoring site to facilitate the access of hairpin ribozymes to their specific target sequences within TAR-containing RNAs.
{"title":"Anchoring hairpin ribozymes to long target RNAs by loop-loop RNA interactions.","authors":"Elena Puerta-Fernández, A. Barroso-deljesús, A. Berzal-Herranz","doi":"10.1089/108729002753670210","DOIUrl":"https://doi.org/10.1089/108729002753670210","url":null,"abstract":"Efficient ribozyme-mediated gene silencing requires the effective binding of a ribozyme to its specific target sequence. Stable stem-loop domains are key elements for efficiency of natural antisense RNAs. This work tests the possibility of using such naturally existing structural motifs for anchoring hairpin ribozymes when targeting long RNAs. Assays were performed with four catalytic antisense RNAs, based on the hairpin ribozyme (HP), that carried a stable stem-loop motif at their 3' end. Extensions consisted of one of the following motifs: the stem-loop II of the natural antisense RNA-CopA, its natural target in CopT, the TAR-RNA motif, or its complementary sequence alphaTAR. Interestingly, the presence of any of these antisense motifs resulted in an enhancement of catalytic performance against the ribozyme's 14-nucleotide-long target RNA (Swt). A series of artificial, long RNA substrates containing the Swt sequence and the natural TAR-RNA stem-loop were constructed and challenged with a catalytic antisense RNA carrying the TAR-complementary stem-loop. This cleaves each of these substrates significantly more efficiently than HP. The deletion of the TAR domain in the substrate, or its substitution by its complementary counterpart alphaTAR, abolishes the positive effect. These results suggest that the enhancement is owed to the interaction of both complementary stem-loop domains. Moreover, they demonstrate that the TAR domain can be used as an anchoring site to facilitate the access of hairpin ribozymes to their specific target sequences within TAR-containing RNAs.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"8 1","pages":"1-9"},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88878182","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-02-01DOI: 10.1089/108729002753670247
J. Bologna, E. Vivés, J. Imbach, F. Morvan
Several lipophilic prodrugs of oligonucleotides (T12 and T20) bearing enzymolabile protecting groups and labeled with fluorescein were synthesized. Their cellular uptake was studied by three different approaches using fluorescence: fluorescence microscopy, flow cytometry and spectrofluorometry. The corresponding prooligonucleotides (pro-oligos) were rapidly and efficiently taken up by HeLa cells and were found homogeneously in the cytoplasm and in the nucleus. The uptake was proportional to their relative lipophilicity and likely proceeded through a passive diffusion mechanism. Uptake followed a dose-response curve. This prooligo approach led to a 2-log increase of uptake in comparison with a T20 phosphorothioate oligonucleotide. Finally, an intracellular concentration of pro-oligo was estimated between 4 and 6 microM for an external concentration of 1 microM and up to 27 microM for an incubation at 10 microM.
{"title":"Uptake and quantification of intracellular concentration of lipophilic pro-oligonucleotides in HeLa cells.","authors":"J. Bologna, E. Vivés, J. Imbach, F. Morvan","doi":"10.1089/108729002753670247","DOIUrl":"https://doi.org/10.1089/108729002753670247","url":null,"abstract":"Several lipophilic prodrugs of oligonucleotides (T12 and T20) bearing enzymolabile protecting groups and labeled with fluorescein were synthesized. Their cellular uptake was studied by three different approaches using fluorescence: fluorescence microscopy, flow cytometry and spectrofluorometry. The corresponding prooligonucleotides (pro-oligos) were rapidly and efficiently taken up by HeLa cells and were found homogeneously in the cytoplasm and in the nucleus. The uptake was proportional to their relative lipophilicity and likely proceeded through a passive diffusion mechanism. Uptake followed a dose-response curve. This prooligo approach led to a 2-log increase of uptake in comparison with a T20 phosphorothioate oligonucleotide. Finally, an intracellular concentration of pro-oligo was estimated between 4 and 6 microM for an external concentration of 1 microM and up to 27 microM for an incubation at 10 microM.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"22 1","pages":"33-41"},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85444284","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-02-01DOI: 10.1089/108729002753670238
L. Papucci, N. Schiavone, M. Donnini, A. Lapucci, E. Luzi, A. Tempestini, E. Witort, S. Capaccioli
Oligodeoxyribonucleotides (ODNs) are currently employed to switch-off genes selectively routinely in the laboratory practice. The drawback of ODN application is that they have been often reported to elicit non-antisense effects by different mechanisms. Recently, it has been shown that double-stranded DNA oligonucleotides (30-mers) with protruding ends activate p53 in a cell-free system. In a previous work, we described that simple addition to the culture medium of heterogeneous DNA combined with cationic lipids culminated in inhibition of mitosis and induction of apoptosis. Here, we report that the same effects are achieved by lipotransfecting cultured cells with phosphorodiester ODNs (30-mers). Such effects of ODN were mediated by a non-antisense mechanism that required the wild-type form of the p53 oncosuppressor protein and was dependent on ODN concentration. Mitosis inhibition and apoptosis induction appeared to be determined by the 3' and 5' free ends of ODNs, which activated p53 independently from their sequence. Most probably, this mechanism is analogous to that evoked by genotoxic agent-induced DNA damage or by lipotransfecting cells with heterogeneous DNA.
{"title":"Phosphodiester oligonucleotides inhibit mitosis and trigger apoptosis by a non-antisense, p53-mediated mechanism.","authors":"L. Papucci, N. Schiavone, M. Donnini, A. Lapucci, E. Luzi, A. Tempestini, E. Witort, S. Capaccioli","doi":"10.1089/108729002753670238","DOIUrl":"https://doi.org/10.1089/108729002753670238","url":null,"abstract":"Oligodeoxyribonucleotides (ODNs) are currently employed to switch-off genes selectively routinely in the laboratory practice. The drawback of ODN application is that they have been often reported to elicit non-antisense effects by different mechanisms. Recently, it has been shown that double-stranded DNA oligonucleotides (30-mers) with protruding ends activate p53 in a cell-free system. In a previous work, we described that simple addition to the culture medium of heterogeneous DNA combined with cationic lipids culminated in inhibition of mitosis and induction of apoptosis. Here, we report that the same effects are achieved by lipotransfecting cultured cells with phosphorodiester ODNs (30-mers). Such effects of ODN were mediated by a non-antisense mechanism that required the wild-type form of the p53 oncosuppressor protein and was dependent on ODN concentration. Mitosis inhibition and apoptosis induction appeared to be determined by the 3' and 5' free ends of ODNs, which activated p53 independently from their sequence. Most probably, this mechanism is analogous to that evoked by genotoxic agent-induced DNA damage or by lipotransfecting cells with heterogeneous DNA.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"73 1","pages":"21-31"},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78098390","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-02-01DOI: 10.1089/108729002753670229
Q. Lau, T. Achenbach, Oliver Borchers, R. Müller, E. Slater
Previously, we have shown that a phosphorothioate antisense oligonucleotide (ODN) targeted against c-raf RNA (ISIS5132; cRaf-AS) induces apoptosis in human tumor cells. We now show that the same ODN also efficiently triggers apoptosis in human tumor xenografts in nu/nu mice. Although cRaf-AS showed a clearly inhibitory effect on the growth of established tumors (approximately 150 mm3) compared to a mismatched control ODN (MM), tumor progression was not prevented. This correlated with a partial refractoriness of the tumor to cRaf-AS-induced cell killing, which seemed to be due to an inhomogeneous and inefficient penetration of the ODN into the tumor tissue rather than cellular resistance. In agreement with this conclusion, we found that growth of small tumors (<50 mm3) was completely inhibited concomitantly with an accumulation of the ODN throughout the tumor. These data show that the cRaf-AS is a highly efficacious antitumor agent, provided accessibility into the tumor tissue is warranted, and suggest that PS-AS-ODN treatment may be particularly useful in an adjuvant setting.
{"title":"In vivo pro-apoptotic and antitumor efficacy of a c-Raf antisense phosphorothioate oligonucleotide: relationship to tumor size.","authors":"Q. Lau, T. Achenbach, Oliver Borchers, R. Müller, E. Slater","doi":"10.1089/108729002753670229","DOIUrl":"https://doi.org/10.1089/108729002753670229","url":null,"abstract":"Previously, we have shown that a phosphorothioate antisense oligonucleotide (ODN) targeted against c-raf RNA (ISIS5132; cRaf-AS) induces apoptosis in human tumor cells. We now show that the same ODN also efficiently triggers apoptosis in human tumor xenografts in nu/nu mice. Although cRaf-AS showed a clearly inhibitory effect on the growth of established tumors (approximately 150 mm3) compared to a mismatched control ODN (MM), tumor progression was not prevented. This correlated with a partial refractoriness of the tumor to cRaf-AS-induced cell killing, which seemed to be due to an inhomogeneous and inefficient penetration of the ODN into the tumor tissue rather than cellular resistance. In agreement with this conclusion, we found that growth of small tumors (<50 mm3) was completely inhibited concomitantly with an accumulation of the ODN throughout the tumor. These data show that the cRaf-AS is a highly efficacious antitumor agent, provided accessibility into the tumor tissue is warranted, and suggest that PS-AS-ODN treatment may be particularly useful in an adjuvant setting.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"1 1","pages":"11-20"},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88206360","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2002-02-01DOI: 10.1089/108729002753670256
O. Sedelnikova, Valeri N. Karamychev, I. Panyutin, Ronald D. Neumann
Triplex-forming oligonucleotides (TFO) are designed to bind sequence specifically to their DNA targets without a significant disturbance of the double helix. They have been proposed to deliver DNA-reactive agents to specific DNA sequences for gene targeting applications. We suggested the use of 125I-labeled TFO for delivery of the energy of radioiodine decay to specific genes. This approach is called antigene radiotherapy. Here we demonstrate the ability of 125I-labeled TFO to produce sequence-specific breaks within a target in the human mdrl gene in cultured cells. TFO and TFO conjugated with a nuclear localization signal peptide (NLS) were delivered into cells using cationic liposomes. This was done either alone or in the presence of an excess of a "ballast" oligonucleotide with an unrelated sequence. In all cases, nuclear localization of TFO and survival of the cells after treatment has been confirmed. Breaks in the gene target were analyzed by restriction enzyme digestion of the DNA recovered from the TFO-treated cells followed by Southern hybridization with DNA probes flanking the target sequence. We have found that TFO/NLS conjugates cleave the target in a concentration-dependent manner regardless of the presence of the "ballast" oligonucleotide. In contrast, TFO without NLS cleaved the target only in the presence of an excess of the "ballast." We hypothesize that TFO and TFO/NLS are delivered into the nucleus by different pathways. These results provide a new insight into the mechanism of intracellular transport of oligonucleotides and open new avenues for improvement of the efficacy of antigene therapies.
{"title":"Sequence-specific gene cleavage in intact mammalian cells by 125I-labeled triplex-forming oligonucleotides conjugated with nuclear localization signal peptide.","authors":"O. Sedelnikova, Valeri N. Karamychev, I. Panyutin, Ronald D. Neumann","doi":"10.1089/108729002753670256","DOIUrl":"https://doi.org/10.1089/108729002753670256","url":null,"abstract":"Triplex-forming oligonucleotides (TFO) are designed to bind sequence specifically to their DNA targets without a significant disturbance of the double helix. They have been proposed to deliver DNA-reactive agents to specific DNA sequences for gene targeting applications. We suggested the use of 125I-labeled TFO for delivery of the energy of radioiodine decay to specific genes. This approach is called antigene radiotherapy. Here we demonstrate the ability of 125I-labeled TFO to produce sequence-specific breaks within a target in the human mdrl gene in cultured cells. TFO and TFO conjugated with a nuclear localization signal peptide (NLS) were delivered into cells using cationic liposomes. This was done either alone or in the presence of an excess of a \"ballast\" oligonucleotide with an unrelated sequence. In all cases, nuclear localization of TFO and survival of the cells after treatment has been confirmed. Breaks in the gene target were analyzed by restriction enzyme digestion of the DNA recovered from the TFO-treated cells followed by Southern hybridization with DNA probes flanking the target sequence. We have found that TFO/NLS conjugates cleave the target in a concentration-dependent manner regardless of the presence of the \"ballast\" oligonucleotide. In contrast, TFO without NLS cleaved the target only in the presence of an excess of the \"ballast.\" We hypothesize that TFO and TFO/NLS are delivered into the nucleus by different pathways. These results provide a new insight into the mechanism of intracellular transport of oligonucleotides and open new avenues for improvement of the efficacy of antigene therapies.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"4 1","pages":"43-9"},"PeriodicalIF":0.0,"publicationDate":"2002-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90162508","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-12-01DOI: 10.1089/108729001753411335
Ramon Güimil García, A. Brank, J. Christman, V. Marquez, R. Eritja
The incorporation of 5-azacytosine residues into DNA causes potent inhibition of DNA (Cytosine-C5) methyltransferases. The synthesis of oligodeoxyribonucleotides incorporating single or multiple 5-aza-2'-deoxycytidine residues at precise sites was undertaken to generate an array of sequences containing the reactive 5-azacytosine base as specific target sites for enzymatic methylation. Preparation of these modified oligonucleotides requires the use of 2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic amino functions. These groups are removed under mild conditions, thus avoiding conventional protocols that are detrimental to the integrity of the 5-azacytosine ring.
{"title":"Synthesis of oligonucleotide inhibitors of DNA (Cytosine-C5) methyltransferase containing 5-azacytosine residues at specific sites.","authors":"Ramon Güimil García, A. Brank, J. Christman, V. Marquez, R. Eritja","doi":"10.1089/108729001753411335","DOIUrl":"https://doi.org/10.1089/108729001753411335","url":null,"abstract":"The incorporation of 5-azacytosine residues into DNA causes potent inhibition of DNA (Cytosine-C5) methyltransferases. The synthesis of oligodeoxyribonucleotides incorporating single or multiple 5-aza-2'-deoxycytidine residues at precise sites was undertaken to generate an array of sequences containing the reactive 5-azacytosine base as specific target sites for enzymatic methylation. Preparation of these modified oligonucleotides requires the use of 2-(p-nitrophenyl)ethyloxycarbonyl (NPEOC) groups for the protection of exocyclic amino functions. These groups are removed under mild conditions, thus avoiding conventional protocols that are detrimental to the integrity of the 5-azacytosine ring.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"150 1","pages":"369-78"},"PeriodicalIF":0.0,"publicationDate":"2001-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75683501","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-12-01DOI: 10.1089/108729001753411344
C. Boiziau, J. Toulmé
In vitro selection is a strategy to identify high-affinity ligands of a predetermined target among a large pool of randomized oligonucleotides. Most in vitro selections are performed with unmodified RNA or DNA sequences, leading to ligands of high affinity and specificity (aptamers) but of very short lifetime in the ex vivo and in vivo context. Only a very limited number of modified triphosphate nucleotides conferring nuclease resistance to the oligomer can be incorporated by polymerases. This encourages the development of alternative methods for the identification of nuclease-resistant aptamers. In this paper, we describe such a method. After selection of 2'O-methyl oligonucleotides against the TAR RNA structure of HIV-1, the complementary DNA sequences are fished out of a pool of randomized oligodeoxynucleotides by Watson-Crick hybridization. The DNA-fished sequences are amplified by PCR as double and single strands, the latter being used to fish back the chemically modified candidates from the initial library. This procedure allows an indirect amplification of the selected candidates. This enriched pool of modified sequences is then used for the next selection round against the target.
体外选择是一种在大量随机寡核苷酸中确定预定目标的高亲和力配体的策略。大多数体外选择是用未经修饰的RNA或DNA序列进行的，这导致了高亲和力和特异性的配体(适体)，但在体内和体外的寿命都很短。只有非常有限数量的修饰的三磷酸核苷酸赋予核酸酶对低聚物的抗性可以被聚合酶结合。这鼓励了鉴定抗核酸酶适体的替代方法的发展。在本文中，我们描述了这种方法。在选择针对HIV-1的TAR RNA结构的2' o -甲基寡核苷酸后，通过沃森-克里克杂交从随机的寡脱氧核苷酸池中取出互补的DNA序列。通过PCR扩增dna序列为双链和单链，后者用于从初始文库中捞回化学修饰的候选序列。这一程序允许间接扩大选定的候选对象。这个经过修改的丰富序列池然后用于针对目标的下一轮选择。
{"title":"A method to select chemically modified aptamers directly.","authors":"C. Boiziau, J. Toulmé","doi":"10.1089/108729001753411344","DOIUrl":"https://doi.org/10.1089/108729001753411344","url":null,"abstract":"In vitro selection is a strategy to identify high-affinity ligands of a predetermined target among a large pool of randomized oligonucleotides. Most in vitro selections are performed with unmodified RNA or DNA sequences, leading to ligands of high affinity and specificity (aptamers) but of very short lifetime in the ex vivo and in vivo context. Only a very limited number of modified triphosphate nucleotides conferring nuclease resistance to the oligomer can be incorporated by polymerases. This encourages the development of alternative methods for the identification of nuclease-resistant aptamers. In this paper, we describe such a method. After selection of 2'O-methyl oligonucleotides against the TAR RNA structure of HIV-1, the complementary DNA sequences are fished out of a pool of randomized oligodeoxynucleotides by Watson-Crick hybridization. The DNA-fished sequences are amplified by PCR as double and single strands, the latter being used to fish back the chemically modified candidates from the initial library. This procedure allows an indirect amplification of the selected candidates. This enriched pool of modified sequences is then used for the next selection round against the target.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"205 1","pages":"379-85"},"PeriodicalIF":0.0,"publicationDate":"2001-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"77093273","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-12-01DOI: 10.1089/108729001753411317
R. Sorrentino, A. Porcellini, D. Spalletti-Cernia, V. Lombari, G. Vecchio, P. Laccetti
Mammalian ras genes encode a family of plasma membrane-bound proteins that function as intermediates in signal transduction pathways involved in cell growth and differentiation. Ras oncogene is frequently involved in neoplastic transformation of different cellular histotypes. In this study, we tested the ability of antisense oligodeoxyribonucleotides (AS-ODN) that have mixed phosphodiester/phosphorothioate backbone, targeted against human N-Ras, to inhibit N-ras gene expression and to specifically interfere with the Ras-dependent activity of mitogen-activated protein kinase (MAPK) in two human cell lines carrying an endogenous N-ras mutated allele at codon 61. Three AS-ODN that inhibit basal MAPK activity have been identified. Moreover, AS-ODN treatment resulted in potent antiproliferative effects in cell culture and great inhibition of N-ras mRNA levels in one of two cell lines. These studies suggest that antisense molecules, targeted against N-Ras, could be of considerable value as a tool to study the N-Ras-specific transduction pathway.
{"title":"Inhibition of MAPK activity, cell proliferation, and anchorage-independent growth by N-Ras antisense in an N-ras-transformed human cell line.","authors":"R. Sorrentino, A. Porcellini, D. Spalletti-Cernia, V. Lombari, G. Vecchio, P. Laccetti","doi":"10.1089/108729001753411317","DOIUrl":"https://doi.org/10.1089/108729001753411317","url":null,"abstract":"Mammalian ras genes encode a family of plasma membrane-bound proteins that function as intermediates in signal transduction pathways involved in cell growth and differentiation. Ras oncogene is frequently involved in neoplastic transformation of different cellular histotypes. In this study, we tested the ability of antisense oligodeoxyribonucleotides (AS-ODN) that have mixed phosphodiester/phosphorothioate backbone, targeted against human N-Ras, to inhibit N-ras gene expression and to specifically interfere with the Ras-dependent activity of mitogen-activated protein kinase (MAPK) in two human cell lines carrying an endogenous N-ras mutated allele at codon 61. Three AS-ODN that inhibit basal MAPK activity have been identified. Moreover, AS-ODN treatment resulted in potent antiproliferative effects in cell culture and great inhibition of N-ras mRNA levels in one of two cell lines. These studies suggest that antisense molecules, targeted against N-Ras, could be of considerable value as a tool to study the N-Ras-specific transduction pathway.","PeriodicalId":7996,"journal":{"name":"Antisense & nucleic acid drug development","volume":"27 1","pages":"349-58"},"PeriodicalIF":0.0,"publicationDate":"2001-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73465448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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