RESUMEN
Poor response to chemotherapy in patients with breast cancer is often associated with overexpression of HER-2/neu. Interference with HER-2 mRNA translation by means of antisense oligonucleotides might improve the efficacy of chemotherapy. To test this hypothesis, eight breast cancer cell lines and a normal human fibroblast cell line were examined for their level of HER-2 expression, their sensitivity to phosphorothioate antisense oligonucleotides (AS HER-2 ODN), and to various chemotherapeutic agents, and the combination of the two. No correlation was found between the intrinsic HER-2 level and either the sensitivity to a particular chemotherapeutic agent alone, or the amount of growth inhibition observed with a specific AS HER-2 ODN concentration. Although sequence specificity and extent of AS HER-2 ODN inhibition of HER-2 synthesis were somewhat higher in the HER-2 overexpressing MDA-MB-453 and SK-BR-3 cells, we found that antisense treatment significantly sensitized all of the breast cancer cells, even MDA-MB-231 and MDA-MB-435 cells, with approximately basal levels of HER-2, to various chemotherapeutic agents. In addition, the combination of AS HER-2 ODN and taxol was shown to synergistically induce apoptosis in MDA-MB-435. These results demonstrate that overexpression of HER-2 would not be a prerequisite for the effective use of AS HER-2 ODN as a combination treatment modality for breast cancer and suggest that the use of AS HER-2 ODN, as part of a combination treatment modality, need not be limited to breast tumors that display elevated levels of HER-2.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Oligonucleótidos Antisentido/farmacología , Receptor ErbB-2/genética , Células Tumorales Cultivadas/efectos de los fármacos , Apoptosis , Western Blotting , Neoplasias de la Mama/metabolismo , División Celular/efectos de los fármacos , Quimioterapia Combinada , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptor ErbB-2/metabolismo , Células Tumorales Cultivadas/metabolismoRESUMEN
The development of aldose reductase inhibitors for the treatment of diabetic complications, such as cataract and retinopathy, has been of intense interest in the pharmaceutical community for the last 20 years. To date, aldose reductase inhibitors have been synthetically developed from leads obtained from in vitro screening studies. Recently, we have observed that mammalian tissues contain intrinsic inhibitors of aldose reductase, which may be used as potential drugs for treating diabetic complications with potentially less side effects than synthetic aldose reductase inhibitors. Intrinsic inhibitor(s) of aldose reductase have been observed in the methanolic extracts from rat and human kidneys and bovine lenses that were subjected to a number of chromatographic techniques, including counter current chromatography, flash chromatography, gel filtration and high pressure liquid chromatography. This inhibition results from a direct interaction between the inhibitor and enzyme. The intrinsic inhibitor, present in the lipophilic fraction of human kidney and bovine lens extracts, can easily penetrate into the lens to inhibit sugar alcohol formation. Intraperitoneal injection of partially purified bovine lens extract inhibited lens polyol formation in young rats fed 50% galactose diet.
Asunto(s)
Aldehído Reductasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Riñón/enzimología , Cristalino/enzimología , Animales , Bovinos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos/aislamiento & purificación , Galactitol/metabolismo , Humanos , Inyecciones Intraperitoneales , Riñón/química , Cristalino/química , Espectroscopía de Resonancia Magnética , Ratas , Ratas Sprague-Dawley , Sorbitol/metabolismoRESUMEN
Activation of the ras oncogene has been implicated in many types of human tumors. It has been shown that downmodulation of ras expression can lead to the reversion of the transformed phenotype of these tumor cells. Antisense oligodeoxyribonucleotides (ODNs) can inhibit gene expression by hybridization to complementary mRNA sequences. To minimize toxicity associated with all-phosphorothioated ODNs and improve cellular uptake, we used partially phosphorothioate (PPS)-modified ODNs having an additional hydrophobic tail at the 3'-end (PPS-C(16)). The PPS ODNs are protected against degradation by PS internucleotide linkages at both the 3'- and 5'-ends and additionally stabilized at internal pyrimidine sites, which are the major sites of endonuclease cleavage. Here we show that anti-ras PPS-C(16) ODN retains the high sequence-specificity of PPS ODNs and provides maximal inhibition of Ras p21 synthesis with minimal toxicity even without the use of a cellular uptake enhancer. Moreover, treatment of T24, a radiation-resistant human tumor cell line that carries a mutant ras gene, with anti-ras PPS-C(16) ODN resulted in a reduction in the radiation resistance of the cells in vitro. We also demonstrate that the growth of RS504 (a human c-Ha-ras transformed NIH/3T3 cell line) mouse tumors was significantly inhibited by the combination of intratumoral injection of anti-ras PPS-C(16) ODN and radiation treatment. These findings indicate the potential of this combination of antisense and conventional radiation therapy as a highly effective cancer treatment modality.
Asunto(s)
Regiones no Traducidas 3'/química , Éteres de Glicerilo/química , Oligorribonucleótidos/farmacología , Tionucleótidos/síntesis química , Tionucleótidos/farmacología , Animales , Codón Iniciador/efectos de los fármacos , Codón Iniciador/metabolismo , Femenino , Genes ras/genética , Humanos , Ratones , Ratones Desnudos , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Oligorribonucleótidos/química , Oligorribonucleótidos Antisentido/metabolismo , Oligorribonucleótidos Antisentido/farmacología , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Proteína Oncogénica p21(ras)/biosíntesis , Fenotipo , ARN Mensajero/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Relación Estructura-Actividad , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
Nucleoside boranophosphates are distinctive in that one of the non-bridging oxygens in the phosphate diester 1 is replaced by a borane moiety (BH3). Although they retain the same net charge, BH3(-)-ODN have unique chemical and biochemical characteristics relative to other analogs. The change in polarity, lipophilicity, nuclease resistance, and the activation of RNase H cleavage of RNA in RNA: boranophosphate hybrids make boranophosphates very attractive for applications in enzymology and molecular biology and as potential antisense agents.
Asunto(s)
Elementos sin Sentido (Genética)/síntesis química , Boranos , Organofosfatos , Compuestos Organofosforados , Tionucleótidos , Secuencia de Bases , Cartilla de ADN , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética/métodos , Estructura Molecular , Reacción en Cadena de la Polimerasa/métodos , Ribonucleasa HRESUMEN
Important chemical and biochemical properties of boranophosphate DNA and RNA oligonucleotides are reviewed. Stereoregular boranophosphate oligomers can be synthesized enzymatically and form stable duplexes with DNA. Fully boronated, non-stereoregular oligothymidylates, synthesized chemically, form hybrids with poly(A) that have lower melting points than oligothymidylate:poly(A), yet they nevertheless can support the RNase H mediated cleavage of RNA.
Asunto(s)
ADN/química , Ácidos Nucleicos/química , Compuestos de Boro/química , Cristalografía por Rayos X , Conformación de Ácido Nucleico , Compuestos Organofosforados/químicaRESUMEN
Modification of the phosphodiester linkages in DNA by replacing one of the nonbridging oxygens with borane, BH3, produces an isoelectronic mimic of DNA called boranophosphates. Nonstereoregular oligodeoxyribonucleoside all-boranophosphates are shown here for the first time to elicit the RNase H hydrolysis of polyribonucleotides. We compared the ability of three types of dodecamers (dodecathymidine phosphate, phosphorothioate, and boranophosphate) to mediate the cleavage of poly(A) by Escherichia coli RNase H1. The rates of poly(A) hydrolysis induced by boranophosphates were 76-fold (at 20 degrees C) and 18-fold (at 30 degrees C) greater than the rates induced by dodecathymidine phosphate. In conjunction with the measured melting temperatures for each heteroduplex, carried out under the same conditions as the RNAse H cleavage experiments, the data establish an inverse relationship between the heteroduplex thermostability and the rate of poly(A) hydrolysis. Chromatographic analysis revealed another correlation: the higher the heteroduplex Tm, the higher the pApA:pApApA ratio in the corresponding hydrolysates. The specific content of these final products provides insight into the relative contribution of RNase H1 exonucleolytic/endonucleolytic mechanisms, with a low ratio for the lower melting heteroduplexes reflecting more endonucleolytic-type hydrolysis. In total, our data support the concept that antisense molecules with a weakened hybridization potential enhance the rate of hydrolysis of RNA in RNA-DNA hybrids.
Asunto(s)
Polirribonucleótidos/metabolismo , Ribonucleasa H/metabolismo , Nucleótidos de Timina/farmacología , Emparejamiento Base , Cationes/farmacología , Cromatografía Líquida de Alta Presión , Escherichia coli/enzimología , Hidrólisis/efectos de los fármacos , Cinética , Imitación Molecular , Ácidos Nucleicos Heterodúplex , Poli A/metabolismo , Solubilidad , Temperatura , Termodinámica , Nucleótidos de Timina/química , Nucleótidos de Timina/metabolismoRESUMEN
DNA-dependent RNA polymerase IIB having a specific activity of 320 u./mg has been isolated from the term placenta homogenate using extraction performed at 4-6 degrees C in the presence of 75 mM ammonium sulfate and 1.5% nonidet P40, fractionation on DEAE-cellulose DE 23, desalting and heparin-agarose chromatography, resulting in 330-fold purification and a 18% yield. Technical details have been determined which are of crucial importance for reproducibility of affinity chromatography. The possibility of proteolysis of the IIc subunit during enzyme purification has been demonstrated.
Asunto(s)
Placenta/enzimología , ARN Polimerasa II/aislamiento & purificación , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Electroforesis en Gel de Poliacrilamida , Humanos , Hidrólisis , ARN Polimerasa II/metabolismo , Reproducibilidad de los ResultadosRESUMEN
RNA polymerase II from human placenta was affinity labelled in crude preparation using two-step technique, which includes treatment of the enzyme with an aldehyde-containing reactive analogue of ATP, ADP or AMP in the presence of poly[d(A-T)] followed (after borohydride reduction) by the elongation of the attached label with [alpha-32P]UTP. A polypeptide of the molecular mass ca. 140 kDa proved to be the labelling target. No labelling was observed in the absence of poly[d(A-T)] or the reagent or in the presence of alpha-amanitin. All the results suggest the attachment of the affinity reagents to the second-largest subunit of the human RNA polymerase II, which therefore takes part in the initiation substrate's binding.
Asunto(s)
Placenta/enzimología , ARN Polimerasa II/química , Marcadores de Afinidad , Sitios de Unión , Cromatografía DEAE-Celulosa , Electroforesis en Gel de Poliacrilamida , Femenino , HumanosAsunto(s)
Astrocitoma/cirugía , Ependimoma/cirugía , Insuficiencia Cardíaca , Prótesis Valvulares Cardíacas , Neoplasias de la Médula Espinal/cirugía , Astrocitoma/complicaciones , Ependimoma/complicaciones , Femenino , Insuficiencia Cardíaca/complicaciones , Humanos , Persona de Mediana Edad , Válvula Mitral , Neoplasias de la Médula Espinal/complicacionesRESUMEN
An analysis of kinetic differences in homopolyribonucleotides hydrolysis by cobra venom endoribonuclease was carried out. It was concluded that the rate and intensity of hydrolysis as well as the length of the linear parts of the kinetic curves are correlated with the content of the nucleotide units with the C3'-endo conformation in the substrates. The structure factor was shown to predominate in some cases over the temperature factor. Protamine sulfate inhibits the enzyme by blocking its phosphodiether bonds. Study on the effects of divalent metal ions demonstrated the possibility that the enzyme-Me2+ complex is functionally active and that the ion-free polyribonucleotides are true substrates.
Asunto(s)
Venenos Elapídicos , Endonucleasas/metabolismo , Endorribonucleasas , Polirribonucleótidos , Ribonucleasas/metabolismo , Animales , Cationes Bivalentes , Cinética , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Poly(G).poly(C) inoculated intravenously to mice in a dose of 100 microgram induced interferon in the blood in amounts comparable to those induced by poly(I).poly (C). In contrast to rapid accumulation (within 2 hours after induction) and rapid disappearance of interferon in response to poly(I).poly(C) inoculation, the interferon induced by poly(G).poly(C) reached the maximum titer by 6 hours and remained at a high level for 24 hours after inoculation. When given to human volunteers intranasally in a dose of 6 mg, the poly(G).poly(C) complex induced interferon in the blood serum in 70% of the subjects in a titer of 85 units/ml within 24 hours.