RESUMO
Although some information is available for specific subsets of miRNAs and several factors have been shown to bind oligonucleotides (ONs), no general transport mechanism for these molecules has been identified to date. In this work, we demonstrate that the nuclear transport of ONs, siRNAs, and miRNAs responds to cellular stress. Furthermore, we have identified a stress-induced response complex (SIRC), which includes Ago-1 and Ago-2 in addition to the transcription and splicing regulators YB1, CTCF, FUS, Smad1, Smad3, and Smad4. The SIRC transports endogenous miRNAs, siRNAs, and ONs to the nucleus. We show that cellular stress can significantly increase ON- or siRNA-directed splicing switch events and endogenous miRNA targeting of nuclear RNAs.
Assuntos
Núcleo Celular/metabolismo , MicroRNAs/metabolismo , Oligonucleotídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Transcrição Gênica/fisiologiaRESUMO
Antisense oligonucleotides (ASOs) are known to trigger mRNA degradation in the nucleus via an RNase H-dependent mechanism. We have now identified a putative cytoplasmic mechanism through which ASO gapmers silence their targets when transfected or delivered gymnotically (i.e. in the absence of any transfection reagent). We have shown that the ASO gapmers can interact with the Ago-2 PAZ domain and can localize into GW-182 mRNA-degradation bodies (GW-bodies). The degradation products of the targeted mRNA, however, are not generated by Ago-2-directed cleavage. The apparent identification of a cytoplasmic pathway complements the previously known nuclear activity of ASOs and concurrently suggests that nuclear localization is not an absolute requirement for gene silencing.
Assuntos
Citoplasma/metabolismo , Inativação Gênica , Oligonucleotídeos Antissenso , Proteínas Argonautas/metabolismo , Linhagem Celular , Citoplasma/química , Técnicas de Transferência de Genes , Oligonucleotídeos Antissenso/análise , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , TransfecçãoRESUMO
BACKGROUND: This multicenter phase II trial evaluated the efficacy and safety of trabectedin in metastatic castration-resistant prostate cancer (CRPC). PATIENTS AND METHODS: Two schedules were evaluated in three cohorts: weekly as 3-h i.v. infusion at 0.58 mg/m(2) for 3 out of 4 weeks (Cohort A, n = 33), and every 3 weeks (q3wk) as 24-h infusion at 1.5 mg/m(2) (Cohort B1, n = 5) and 1.2 mg/m(2) (Cohort B2, n = 20). The primary end point was prostate-specific antigen (PSA) response; secondary end points included safety, tolerability and time to progression (TTP). RESULTS: Trabectedin resulted in PSA declines ≥ 50% in 12.5% (Cohort A) and 10.5% (Cohort B2) of patients. Among men pretreated with taxane-based chemotherapy, PSA response was 13.6% (Cohort A) and 15.4% (Cohort B2). PSA responses lasted 4.1-8.6 months, and median TTP was 1.5 months (Cohort A) and 1.9 months (Cohort B2). The dose of 1.5 mg/m(2) (approved for soft tissue sarcoma) given as 24-h infusion q3wk was not tolerable in these patients. At 1.2 mg/m(2) q3wk and 0.58 mg/m(2) weekly, the most common adverse events were nausea, fatigue and transient neutropenia and transaminase increase. CONCLUSIONS: Two different trabectedin schedules showed modest activity in metastatic CRPC. Further studies may require identification of predictive factors of response in prostate cancer.
Assuntos
Dioxóis/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Sarcoma/tratamento farmacológico , Tetra-Hidroisoquinolinas/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Alquilantes/efeitos adversos , Antineoplásicos Alquilantes/uso terapêutico , Estudos de Coortes , Dioxóis/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Orquiectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Sarcoma/patologia , Sarcoma/cirurgia , Tetra-Hidroisoquinolinas/efeitos adversos , Trabectedina , Falha de Tratamento , Resultado do TratamentoRESUMO
For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.
Assuntos
Inativação Gênica , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos/administração & dosagem , Animais , Linhagem Celular Tumoral , Humanos , Indicadores e Reagentes , Camundongos , Oligonucleotídeos/análise , Oligonucleotídeos Antissenso/análise , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , TransfecçãoRESUMO
We have studied the interactions of phosphodiester and phosphorothioate oligodeoxynucleotides with Mac-1 (CD11b/CD18; alpha M beta 2), a heparin-binding integrin found predominantly on the surface of polymorphonuclear leukocytes (PMNs), macrophages and natural killer cells. Binding of a homopolymer of thymidine occurred on both the alpha M and beta 2 subunits. Soluble fibrinogen, a natural ligand for Mac-1, was an excellent competitor of the binding of a phosphorothioate oligodeoxynucleotide to both TNF-alpha-activated and nonactivated PMNs. Upregulation of cell-surface Mac-1 expression increased cell-surface binding of oligodeoxynucleotides. Binding was inhibited by anti-Mac-1 monoclonal antibodies, and the increase in cell-surface binding was correlated with a three- to fourfold increase in internalization by PMNs. An oligodeoxynucleotide inhibited beta 2-dependent migration through Matrigel, but the production of reactive oxygen species in PMNs adherent to fibrinogen dramatically increased. Thus, our data demonstrate that Mac-1 is a cell-surface receptor for oligodeoxynucleotides that can mediate their internalization and that this binding may have important functional consequences.
Assuntos
Antígenos CD18/metabolismo , Proteínas de Ligação a DNA/metabolismo , Antígeno de Macrófago 1/metabolismo , Proteínas de Membrana , Neutrófilos/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Receptores de Lipoproteínas , Anticorpos Monoclonais/farmacologia , Ligação Competitiva , Antígenos CD18/genética , Antígenos CD18/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Humanos , Ligantes , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/imunologia , Ligação Proteica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptores Imunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores Classe B , Transdução de Sinais , Tionucleotídeos/metabolismo , Regulação para CimaRESUMO
Defibrotide (DF) is a mixture of porcine-derived single-stranded phosphodiester oligonucleotides (9-80-mer; average, 50-mer) that has been successfully used to treat severe hepatic veno-occlusive disease (sVOD) with multiorgan failure (MOF) in patients who have received cytotoxic chemotherapy in preparation for bone marrow transplantation. However, its mechanism of action is unknown. Herein, we show that DF and phosphodiester oligonucleotides can bind to heparin-binding proteins (eg, basic fibroblast growth factor [bFGF] but not vascular endothelial growth factor [VEGF] 165) with low nanomolar affinity. This binding occurred in a length- and concentration-dependent manner. DF can mobilize proangiogenic factors such as bFGF from their depot or storage sites on bovine corneal endothelial matrix. However, these molecules do not interfere with high-affinity binding of bFGF to FGFR1 IIIc but can replace heparin as a required cofactor for binding and hence cellular mitogenesis. DF also protects bFGF against digestion by trypsin and chymotrypsin and from air oxidation. In addition, DF binds to collagen I with low nanomolar affinity and can promote human microvascular endothelial cell-1 (HMEC-1) cell mitogenesis and tubular morphogenesis in three-dimensional collagen I gels. Thus, our data suggest that DF may provide a stimulus to the sinusoidal endothelium of a liver that has suffered a severe angiotoxic event, thus helping to ameliorate the clinical sVOD/MOF syndrome.
Assuntos
Células Endoteliais/metabolismo , Fibrinolíticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Hepatopatia Veno-Oclusiva/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Polidesoxirribonucleotídeos/farmacologia , Animais , Transplante de Medula Óssea/efeitos adversos , Bovinos , Linhagem Celular Transformada , Córnea/metabolismo , Córnea/patologia , Células Endoteliais/patologia , Fibrinolíticos/uso terapêutico , Hepatopatia Veno-Oclusiva/tratamento farmacológico , Hepatopatia Veno-Oclusiva/etiologia , Hepatopatia Veno-Oclusiva/patologia , Humanos , Insuficiência de Múltiplos Órgãos/tratamento farmacológico , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/metabolismo , Insuficiência de Múltiplos Órgãos/patologia , Polidesoxirribonucleotídeos/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: We examined the effects of G3139 on the interaction of heparin-binding proteins [e.g., fibroblast growth factor 2 (FGF2) and collagen I] with endothelial cells. G3139 is an 18-mer phosphorothioate oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA. A randomized, prospective global phase III trial in advanced melanoma (GM301) has evaluated G3139 in combination with dacarbazine. However, the mechanism of action of G3139 is incompletely understood because it is unlikely that Bcl-2 silencing is the sole mechanism for chemosensitization in melanoma cells. EXPERIMENTAL DESIGN: The ability of G3139 to interact with and protect heparin-binding proteins was quantitated. The effects of G3139 on the binding of FGF2 to high-affinity cell surface receptors and the induction of cellular mitogenesis and tubular morphogenesis in HMEC-1 and human umbilical vascular endothelial cells were determined. RESULTS: G3139 binds with picomolar affinity to collagen I. By replacing heparin, the drug can potentiate the binding of FGF2 to FGFR1 IIIc, and it protects FGF from oxidation and proteolysis. G3139 can increase endothelial cell mitogenesis and tubular morphogenesis of HMEC-1 cells in three-dimensional collagen gels, increases the mitogenesis of human umbilical vascular endothelial cells similarly, and induces vessel sprouts in the rat aortic ring model. CONCLUSIONS: G3139 dramatically affects the behavior of endothelial cells. There may be a correlation between this observation and the treatment interaction with lactate dehydrogenase observed clinically.
Assuntos
Células Endoteliais/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Tionucleotídeos/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Becaplermina , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Humanos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-sis , Ratos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/efeitos dos fármacos , Tionucleotídeos/genética , Tionucleotídeos/metabolismoRESUMO
Because of the specificity of Watson-Crick base pairing, attempts are now being made to use oligodeoxynucleotides (oligos) in the therapy of human disease. However, for a successful outcome, the oligo must meet at least six criteria: (i) the oligos can be synthesized easily and in bulk; (ii) the oligos must be stable in vivo; (iii) the oligos must be able to enter the target cell; (iv) the oligos must be retained by the target cell; (v) the oligos must be able to interact with their cellular targets; and (vi) the oligos should not interact in a non-sequence-specific manner with other macromolecules. Phosphorothioate oligos are examples of oligos that are being considered for clinical therapeutic trials and meet some, but not all, of these criteria. The potential use of phosphorothioate oligos as inhibitors of viral replication is highlighted.
Assuntos
Antivirais/uso terapêutico , Oligonucleotídeos Antissenso/uso terapêutico , Biossíntese de Proteínas/efeitos dos fármacos , Tionucleotídeos/uso terapêutico , Animais , Antivirais/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Herpesviridae/efeitos dos fármacos , Herpesviridae/fisiologia , Humanos , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Tionucleotídeos/farmacologia , Células Tumorais Cultivadas , Replicação Viral/efeitos dos fármacosAssuntos
Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/tratamento farmacológico , Carcinoma Adrenocortical/secundário , Antineoplásicos Hormonais/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Mitotano/administração & dosagem , Neoplasias do Córtex Suprarrenal/cirurgia , Adrenalectomia , Esquema de Medicação , Feminino , Humanos , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons , Fatores de Tempo , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
Phosphorothioate oligodeoxynucleotides (PS oligos) are antisense (sequence-specific) inhibitors of vascular smooth muscle cell (SMC) proliferation when targeted against different genes. Recently an aptameric G-quartet inhibitory effect of PS oligos has been demonstrated. To determine whether PS oligos manifest non-G-quartet, non-sequence-specific effects on human aortic SMC, we examined the effects of S-dC28, a 28-mer phosphorothioate cytidine homopolymer, on SMC proliferation induced by several SMC mitogens. S-dC28 significantly inhibited SMC proliferation induced by 10% FBS as well as the mitogens PDGF, bFGF, and EGF without cytotoxicity. Moreover, S-dC28 abrogated PDGF-induced in vitro migration in a modified micro-Boyden chamber. Furthermore, S-dC28 manifested in vivo antiproliferative effects in the rat carotid balloon injury model. S-dC28 suppressed neointimal cross-sectional area by 73% and the intima/media area ratio by 59%. Therefore, PS oligos exert potent non-G-quartet, non-sequence-specific effects on in vitro SMC proliferation and migration as well as in vivo neointimal formation.
Assuntos
Substâncias de Crescimento/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Tionucleotídeos , Túnica Íntima/efeitos dos fármacos , Animais , Aorta , Sequência de Bases , Becaplermina , Calorimetria , Artérias Carótidas/efeitos dos fármacos , Lesões das Artérias Carótidas , Cateterismo/efeitos adversos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Dexametasona/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , L-Lactato Desidrogenase , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Neovascularização Fisiológica/fisiologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Túnica Íntima/citologia , Túnica Íntima/fisiologia , Túnica Média/citologia , Túnica Média/efeitos dos fármacos , Túnica Média/fisiologiaRESUMO
PURPOSE: Bcl-2 is an apoptotic protein that is highly expressed in advanced melanoma. Several strategies have been employed to target the expression of this protein, including G3139, an 18-mer phosphorothioate oligodeoxyribonucleotide targeted to the initiation region of the Bcl-2 mRNA. This compound has recently completed phase III global clinical evaluation, but the function of Bcl-2 as a target in melanoma has not been completely clarified. To help resolve this question, we have permanently and stably down-regulated Bcl-2 protein and mRNA expression in 518A2 cells by two different technologies and evaluated the resulting clones both in vitro and in vivo. EXPERIMENTAL DESIGN: 518A2 melanoma cells were transfected with plasmids engineered to produce either a single-stranded antisense oligonucleotide targeted to the initiation codon region of the Bcl-2 mRNA or a short hairpin RNA also targeted to the Bcl-2 mRNA. In vitro growth, the apoptotic response to G3139, and the G3139-induced release of cytochrome c from isolated mitochondria were evaluated. Cells were then xenografted into severe combined immunodeficient mice and tumor growth was measured. RESULTS: In vitro, down-regulation of Bcl-2 expression by either method produced no change either in the rate of growth or in sensitivity to standard cytotoxic chemotherapeutic agents. Likewise, the induction of apoptosis by G3139 was entirely Bcl-2 independent. In addition, the G3139-induced release from isolated mitochondria was also relatively independent of Bcl-2 expression. However, when xenografted into severe combined immunodeficient mice, cells with silenced Bcl-2, using either technology, either failed to grow at all or grew to tumors of low volume and then completely regressed. In contrast, control cells with "normal" levels of Bcl-2 protein expression expanded to be large, necrotic tumors. CONCLUSIONS: The presence of Bcl-2 protein profoundly affects the ability of 518A2 melanoma cells to grow as human tumor xenografts in severe combined immunodeficient mice. The in vivo role of Bcl-2 in melanoma cells thus differs significantly from its in vitro role, and these experiments further suggest that Bcl-2 may be an important therapeutic target even in tumors that do not contain the t14:18 translocation.
Assuntos
Melanoma/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/genética , Sequência de Bases , Citocromos c/metabolismo , DNA Antissenso/genética , Regulação para Baixo , Inativação Gênica , Humanos , Melanoma/metabolismo , Melanoma/patologia , Melanoma/terapia , Camundongos , Camundongos Endogâmicos ICR , Camundongos SCID , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Tionucleotídeos/genética , Tionucleotídeos/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Endorribonucleases/metabolismo , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/genética , Western Blotting , Regulação para Baixo , Ativação Enzimática , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Conformação de Ácido Nucleico , Oligorribonucleotídeos/química , Oligorribonucleotídeos/genética , Fosfatidiletanolaminas/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Proteínas Proto-Oncogênicas c-bcl-2/análise , Proteínas Proto-Oncogênicas c-bcl-2/genética , Processamento Pós-Transcricional do RNA/genética , RNA Antissenso/química , RNA Antissenso/genética , RNA Antissenso/fisiologia , RNA Mensageiro/genética , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribonuclease H/metabolismo , Ribonuclease P , Especificidade por Substrato , Transfecção/métodos , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Proteína bcl-X , Pequeno RNA não TraduzidoRESUMO
G3139 is a phosphorothioate oligodeoxyribonucleotide that is targeted to the initiation codon region of the Bcl-2 mRNA, which downregulates Bcl-2 protein and mRNA expression via an antisense mechanism. In previous work, we have demonstrated that the phenotype observed in several prostate and melanoma cell lines after treatment with G3139 appears to be Bcl-2 independent. In contrast, downregulation of Bcl-2 expression by a small interfering RNA (siRNA) produced little or no phenotype change. In the present work, we performed an Agilent oligonucleotide microarray assay on mRNA isolated from PC3 prostate cancer cells that were treated with two different oligonucleotide gene-silencing reagents. G3139 and a Bcl-2-targeting siRNA both downregulate Bcl-2 expression, but via different mechanisms. A side-by-side comparative analysis showed that the expression profile generated by these molecules differs substantially. The study revealed upregulation of the expression of stress-inducible genes in PC3 cells at 1 and 3 days after a 5-h transfection with G3139 complexed with Lipofectamine 2000. In contrast, only a very diminished stress response was seen 1 and 3 days after a 24-h transfection of siRNA/Lipofectamine 2000 complexes. These results highlight the profound differences in off-target effects in cells treated with the phosphorothioate oligonucleotide G3139 and with an siRNA targeted to the same gene, and provide further evidence that the mechanism of action of G3139 is not related to Bcl-2 silencing.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Interferente Pequeno/metabolismo , Tionucleotídeos/genética , Linhagem Celular Tumoral , Proliferação de Células , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Tionucleotídeos/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
Veno-occlusive disease of the liver (VOD) remains a troubling and potentially fatal complication of high-dose chemotherapy and hematopoietic stem cell transplantation conditioning regimens. No effective therapy has been available for these patients to date, and the best supportive care measures remain woefully inadequate. Defibrotide (DF) (Gentium, S.p.A., Como, Italy), a polydisperse mixture of all the single-stranded phosphodiester oligodeoxyribonucleotides that can be obtained from the controlled depolymerization of porcine intestinal mucosal genomic DNA, seems to offer a safe and effective treatment for some patients suffering from severe VOD, a condition for which no accepted standard therapy currently exists. Early clinical studies evaluating the efficacy of DF for the treatment of severe VOD in patients undergoing hematopoietic stem cell transplantation have been very encouraging. Approximately 45% of the patients treated in multiple initial phase II clinical trials achieved a complete response at day +100, demonstrating normalization of serum bilirubin and resolution of the clinical syndrome. However, although multi-institutional, these represented single arm studies. A large, FDA-approved, pivotal, prospective, multi-institutional, global phase III trial of DF vs. historical controls (best available therapy) commenced in the first quarter of 2006 and should provide further validation of DF's efficacy. The drug seems to have few significant side effects, and almost all test subjects who have received this treatment have tolerated it well. Although the mechanism of action remains unclear, the drug exerts minimal systemic anticoagulant effects yet appears to induce numerous antithrombotic and profibrinolytic effects both in vitro and in vivo. It may function as an adenosine receptor agonist and causes increased concentrations of endogenous prostaglandins, which modulate thrombomodulin, platelets, and fibrinolysis. It also appears to block lipopolysaccharide (LPS)-induced tissue factor (TF) expression. However, despite the fact the DF is composed of oligonucleotides, its mechanism of action, which at the present time is unclear, is not related to Watson-Crick base pair-dependent downregulation of gene expression but is rather likely a result of its polyanionic nature.
Assuntos
Hepatopatia Veno-Oclusiva/tratamento farmacológico , Polidesoxirribonucleotídeos/uso terapêutico , Hepatopatia Veno-Oclusiva/diagnóstico , Hepatopatia Veno-Oclusiva/fisiopatologia , Humanos , Polidesoxirribonucleotídeos/farmacologia , Prognóstico , Resultado do TratamentoRESUMO
Several phosphorothioate antisense oligodeoxynucleotides (ODN) are developed to target factors potentially involved in tumor growth and apoptosis suppression. Among them, the 18-mer G3139 (Oblimersen), which targets Bcl-2, is currently being tested in phase II and phase III clinical trials for various tumors in combination with chemotherapy. On the other hand, ODNs containing CpG dinucleotides (CpG-ODN) within specific-sequence contexts (CpG motifs) have been shown to activate rodent or primate immune cells via toll-like receptor 9 (TLR9) and have demonstrated remarkable T cell-dependent antitumor efficacy in a series of murine tumor models. However, immune cell activation by CpG-ODN is largely diminished upon C-5 methylation at CpG cytosine. As G3139 contains CpG motifs, we questioned whether the antitumor effects seen in human tumor xenografts might be abrogated by cytosine C-5 methylation of G3139, which retained the ability of G3139 to suppress Bcl-2 expression in tissue culture, or by similar derivatization of other phosphorothioate ODNs developed for the immune activation of rodent or human cells. The in vivo antitumor efficacy of the immunostimulatory H1826 and H2006 ODNs was compared with that of G3139. Bcl-2 suppression achieved by G3139 purportedly sensitizes tumor cells toward cytotoxic agents, and some of the experiments employed combinations of ODN with such drugs as cisplatin or etoposide. H1826, H2006, and G3139 all produced similar, striking, growth inhibitory effects on either H69 SCLC, A2780 ovarian carcinoma, or A549 lung adenocarcinoma human tumor xenografts at doses of 0.3 mg/kg and 1 mg/kg (H1826, H2006) or 12 mg/kg (G3139) per day. In contrast, the H2006-mC (1 mg/kg) or G3139-mC (12 mg/kg) derivatives demonstrated no significant antitumor effects. The combination of G3139 (12 mg/kg) with cisplatin produced some additive antitumor efficacy, which was not seen in combinations of G3139-mC (12 mg/kg) or H1826 (1 mg/kg) with cisplatin. G3139, at a dose of 12 mg/kg, alone induced extensive enlargement of the spleen. Immunostimulation was evaluated in vitro by flow cytometric measurements of the CD80 and CD86 activation markers found on CD19+ murine splenocytes. The CpG-ODN producing strong antitumor effects in vivo also induced these activation markers in vitro, in contrast to the in vivo inactive G3139-mC. Our data indicate a significant contribution of the immunostimulatory properties of CpG-ODN (including G3139) to the antitumor effects observed in nude mouse xenograft models. This is in contrast to previous data presented by other authors indicating that the activity of G3139 in human tumor xenografts was Bcl-2 specific. Furthermore, as nude mice are devoid of T cells, a T cell-mediated immune response apparently is not required for the potent antitumor responses observed here; innate immune responses are sufficient.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Oligodesoxirribonucleotídeos/uso terapêutico , Tionucleotídeos/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linfócitos B/efeitos dos fármacos , Carcinoma/tratamento farmacológico , Carcinoma/imunologia , Linhagem Celular Tumoral , Citosina/química , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Ativação Linfocitária , Metilação , Camundongos , Camundongos Nus , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/química , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tionucleotídeos/administração & dosagem , Tionucleotídeos/química , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Oligonucleotídeos Antissenso/farmacologia , Tionucleotídeos/farmacologia , Sequência de Bases , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , Polieletrólitos , Polímeros/farmacologia , Ligação Proteica , Projetos de Pesquisa , Tionucleotídeos/química , Tionucleotídeos/metabolismoRESUMO
Recent studies have suggested that loss of heterozygosity at loci on the short arm of human chromosome 11 (11p) may be important in the pathogenesis of benign and malignant adrenal cortical tumors. To test this concept, adrenocortical carcinomas from nine patients and benign adrenal cortical lesions from eight patients were tested for loss of alleles at loci on human chromosomes 11, 13, and 17. All patients with adrenocortical carcinoma whose normal somatic tissues were heterozygous for a locus on chromosome 17p had lost alleles in the tumor. Four of six patients with adrenocortical carcinoma who were heterozygous for one or more alleles on chromosome 11p in normal tissues had lost 11p alleles in the tumor. Three of six patients with adrenocortical carcinoma showed loss of 13q alleles in the tumor. Loss of alleles on chromosomes 11p, 13q, and 17p was observed in primary tumors and metastases but not in adrenocortical adenomas or hyperplastic lesions of the adrenal cortex. One patient with adrenocortical carcinoma had a somatic mutation in the HRAS1 gene in the normal adrenal gland. The consistency of the genetic changes on chromosomes 11p, 13q, and 17p suggests that they are important in the pathogenesis of adrenocortical carcinoma.
Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma/genética , Deleção Cromossômica , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 17 , Glândulas Suprarrenais/análise , Alelos , DNA/isolamento & purificação , Humanos , Fígado/análise , Pulmão/análise , Miocárdio/análise , Polimorfismo de Fragmento de RestriçãoRESUMO
The past several years have seen an explosive growth in the application of antisense oligodeoxynucleotides as modulators of gene expression. In this review, we discuss the various classes of compounds currently receiving attention. These include unmodified oligodeoxynucleotides, methylphosphonates, phosphorothioates, and alpha-oligonucleotides. The relative advantages and disadvantages of each class with regard to parameters such as (a) duplex stability (b) nuclease resistance, and (c) effectiveness of specific gene inhibition are noted. Oligodeoxynucleotides covalently linked to intercalating and other reactive groups are also described, as are possible mechanisms of oligodeoxynucleotide action. Finally, we consider potential future directions for this field and note that it holds the promise for specific gene therapy.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Antivirais/farmacologia , Humanos , Neoplasias/tratamento farmacológico , Oligodesoxirribonucleotídeos/uso terapêutico , Compostos Organofosforados/farmacologia , Relação Estrutura-AtividadeRESUMO
Both Bcl-xL and Bcl-2, antiapoptotic members of the Bcl family, are found in prostate cancer cell lines. Although these proteins may have similar antiapoptotic functions, it is not clear to what extent each serves as an antiapoptotic effector in prostate cancer cells. We engineered LNCaP and PC-3 cells to overexpress Bcl-xL protein and demonstrated that this desensitized them to the effects of cytotoxic chemotherapy. We then used two "antisense" strategies to down-regulate Bcl-xL protein expression in the parental lines. The first strategy used CS-propynylated phosphorothioate-phosphodiester oligonucleotides and co-down-regulated both Bcl-xL and Bcl-2; the second strategy used isosequential "gap-mer" phosphorothioate oligonucleotides containing 2'-O-methyl oligoribonucleotides at the 3' and 5' termini. In this case, only Bcl-xL protein expression was affected. The most active oligonucleotides of both types decreased the level of Bcl-xL protein expression to 5-30% of the control level. Multiple controls were inactive. Experiments combining oligonucleotide treatment with cytotoxic chemotherapeutic agents (paclitaxel, docetaxel, etoposide, vinblastine, carboplatin, and mitoxantrone) demonstrated a marked increase in the sensitivity of these prostate cancer cells. However, the increase in chemosensitivity in PC-3 cells was statistically identical (except mitoxantrone) for both "antisense" strategies, indicating that basal expression of Bcl-2, in contrast to that of Bcl-xL, may play little cytoprotective role in these cells.
Assuntos
Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Fenótipo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2 , Proteína bcl-XRESUMO
bcl-xL is a M(r) 26,000 bcl-2 homologue that is highly expressed in prostate cancer cells. In previous studies, the down-regulation of its expression by antisense oligonucleotides led to resistance. In this work, the 445-bp 5' terminus of the bcl-xL cDNA was cloned in the antisense orientation and stably transfected into DU145 and LNCaP prostate cancer cells. In the DU145 (and to a lesser extent the LNCaP) transfectants, phenotypic changes (versus mock-transfected cells) included an increase in doubling time (from 36 to 175 h) in the clone in which bcl-xL protein expression was 25% of control. The transfectants did not demonstrate characteristic apoptotic changes, as demonstrated by 4',6-diamidino-2-phenylindole staining, lack of either DNA laddering, caspase-3 activation, or poly(ADP)ribose and lamin cleavage, and the absence of a significant sub-G(0) population. Cell cycle analysis demonstrated an increase in a tetraploid population (from 28% to 66%), as well as the appearance of a hypertetraploid population. Levels of cIAP-1 protein were almost undetectable in the mock cells but increased at least 25-fold in the DU145 transfectants. The down-regulation of bcl-xL in both DU145 (and to a much lesser extent in LNCaP) cells led to their resistance to cytotoxic agents, including docetaxel, mitoxantrone, etoposide, vinblastine, and carboplatin. Reversion of bcl-xL expression in stable DU145 transfectants to nearly the levels found in the mock-transfected cells was accomplished by retroviral infection of the cells with a bcl-xL sense cDNA under control of a prolific promoter. This led to a dramatic increase in the growth rate and in BrdUrd incorporation, as well as a sharp decrease in the expression of cIAP-1 protein. Overall, these findings highlight the adaptability of prostate cancer cells to loss of bcl-xL and suggest that in addition to its prosurvival role, bcl-xL protein may also be involved in the regulation of the rate of cellular proliferation.