RESUMEN
We have previously identified phosphodiester oligonucleotides exclusively made of G and T bases, named GTn, that significantly inhibit human cancer cell growth and recognize specific nuclear single-stranded DNA binding proteins. We wished to examine the ability of the modified GTn oligonucleotides with different degrees of phosphorothioate modifications to bind specifically to the same nuclear proteins recognized by the GTn phosphodiester analogues and their cytotoxic effect on the human T-lymphoblastic CCRF-CEM cell line. We showed that the full phosphorothioate GTn oligonucleotide was neither able to specifically recognize those nuclear proteins, nor cytotoxic. In contrast, the 3'-phosphorothioate-protected GTn oligonucleotides can maintain the specific protein-binding activity. The end-modified phosphorothioate oligonucleotides were also able to elicit the dose-dependent cell growth inhibition effect, but a loss in the cytotoxic ability was observed increasing the extent of sulphur modification of the sequences. Our results indicate that phosphorothioate oligonucleotides directed at specific single-stranded DNA-binding proteins should contain a number of phosphorothioate end-linkages which should be related to the length of the sequence, in order to maintain the same biological activities exerted by their phosphodiester analogues.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Leucemia-Linfoma de Células T del Adulto/metabolismo , Proteínas de Neoplasias/metabolismo , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Secuencia de Bases , División Celular , ADN de Neoplasias/metabolismo , ADN de Cadena Simple/metabolismo , Humanos , Leucemia-Linfoma de Células T del Adulto/genética , Leucemia-Linfoma de Células T del Adulto/patología , Oligodesoxirribonucleótidos/genética , Tionucleótidos/química , Tionucleótidos/genética , Tionucleótidos/metabolismo , Células Tumorales CultivadasRESUMEN
We have identified phosphodiester oligonucleotides composed of G and T bases, named GTn, which are able to inhibit the cellular growth of human cancer cell lines by recognising specific nuclear proteins. We demonstrated that GTn oligonucleotides require a length of at least 20 nucleotides in order to exert a significant cytotoxic effect and to retain the specific protein binding ability. In addition, we found that GTn cytotoxicity was lost when A or C bases were introduced at either 3' and 5' end or within the GTn sequences.
Asunto(s)
Proteínas Nucleares/metabolismo , Oligonucleótidos/metabolismo , Biopolímeros , Humanos , Células Tumorales CultivadasRESUMEN
We have demonstrated previously that the GT triplex-forming oligodeoxyribonucleotide (TFO) d(TGTGTTTTTGTTTTGTTGGTTTTGTTT), named TFO ID, targeted to a polypyrimidine-polypurine coding sequence located within human multidrug-resistance mdrl gene, specifically and significantly reduced mdrl mRNA levels in the drug-resistant T-leukemic CEM-VLB100 cell line. In this article, we demonstrate that TFO 1D is effective at inhibiting not only transcription but also replication of mdrl genes, leading to a loss of amplified gene copies in the drug-resistant colon adenocarcinoma LoVo DX cell line. In contrast, TFO ID does not alter replication of the constitutive mdrl gene copy in the corresponding parental sensitive LoVo 109 cell line. A specific reduction in mdrl gene amplification levels was also obtained with the pyrimidine TFO d(CTTTTTCTTTTCTTCCTTTTCTTT), named TFO 24TC, directed against the same polypyrimidine-polypurine sequence of the mdrl gene. We suggest that triple helix-forming oligonucleotides might affect the replication of unstable chromosomal elements as amplicons in actively replicating cells by causing a local impairment of DNA polymerase activity. This study lends support to the notion that TFO may be used to reduce gene amplification aiming to control neoplastic progression in cancer cells bearing amplified oncogenes.
Asunto(s)
ADN/biosíntesis , Resistencia a Múltiples Medicamentos/genética , Genes MDR , Oligodesoxirribonucleótidos/farmacología , Secuencia de Bases , Southern Blotting , Humanos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
Oligonucleotides can specifically target not only nucleic acids but also proteins. Some proteins recognizing oligonucleotides in a sequence-specific manner have been related to cancer transformation and progression. We have found that oligonucleotides composed by repeated and/or variable intervals of GTn with 1 < or = n < or = 7, are able to exert a specific and dose-dependent growth inhibition on human CCRF-CEM, CEM-VLB300, U937, Jurkat, H9 and HeLa tumor cell lines. In contrast, G-->C, G-->A, T-->C and T-->A base substituted control oligonucleotides do not significantly alter cellular growth. In all cell lines, a nuclear protein (molecular mass = 45+/-7 kDa), which specifically recognizes GTn, was identified. Our hypothesis is that the formation of the GTn-protein complex in human cancer cell lines may be involved in the growth inhibition effect. In fact, we found that the reduction or lack of cytotoxic effects by GTn in phorbol 12-myristate 13-acetate-treated CCRF-CEM cells and in normal human lymphocytes is paralleled by the simultaneous reduction or lack of GTn-protein complex. Oligonucleotides specifically 'quenching' intracellular protein activities by forming oligonucleotide-protein complexes may be of potential interest in the treatment of human tumors.
Asunto(s)
División Celular/efectos de los fármacos , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Oligodesoxirribonucleótidos/farmacología , Supervivencia Celular/efectos de los fármacos , Reactivos de Enlaces Cruzados/metabolismo , Citoplasma/química , Proteínas de Unión al ADN/análisis , Humanos , Interleucina-2/farmacología , Linfocitos/efectos de los fármacos , Proteínas Nucleares/análisis , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/farmacocinética , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/análisis , Secuencias Repetitivas de Ácidos Nucleicos/genética , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
The human mdr1 gene encodes a transmembrane glycoprotein the over-expression of which is associated with development of multidrug resistance in human tumor cells. A negative modulation of human mdr1 has been attempted via a 27-mer unmodified triple helix-forming oligonucleotide, named 1D, targeted to a homopurine sequence in the coding region of the gene. By administering 10 microM of 1D we could find a significant reduction in MDR1 mRNA levels in the human drug-resistant cell line CEM-VLB100. This effect appears to be specific and due to a transient block of RNA polymerase mediated by triple helix formation.