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1.
EMBO J ; 32(16): 2248-63, 2013 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-23860128

RESUMO

Squamous cell carcinomas (SCCs) are highly heterogeneous tumours, resulting from deranged expression of genes involved in squamous cell differentiation. Here we report that microRNA-34a (miR-34a) functions as a novel node in the squamous cell differentiation network, with SIRT6 as a critical target. miR-34a expression increases with keratinocyte differentiation, while it is suppressed in skin and oral SCCs, SCC cell lines, and aberrantly differentiating primary human keratinocytes (HKCs). Expression of this miRNA is restored in SCC cells, in parallel with differentiation, by reversion of genomic DNA methylation or wild-type p53 expression. In normal HKCs, the pro-differentiation effects of increased p53 activity or UVB exposure are miR-34a-dependent, and increased miR-34a levels are sufficient to induce differentiation of these cells both in vitro and in vivo. SIRT6, a sirtuin family member not previously connected with miR-34a function, is a direct target of this miRNA in HKCs, and SIRT6 down-modulation is sufficient to reproduce the miR-34a pro-differentiation effects. The findings are of likely biological significance, as SIRT6 is oppositely expressed to miR-34a in normal keratinocytes and keratinocyte-derived tumours.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Diferenciação Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Queratinócitos/fisiologia , MicroRNAs/metabolismo , Sirtuínas/metabolismo , Diferenciação Celular/efeitos da radiação , Primers do DNA/genética , Humanos , Queratinócitos/metabolismo , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo Real , Receptor Notch1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta
2.
Nat Clin Pract Endocrinol Metab ; 3(12): 827-34, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18026161

RESUMO

The capacity of tiny, noncoding RNA molecules (including small, interfering RNA molecules and micro-RNA molecules [miRNAs]) to control gene expression in a very specific and efficient manner has opened new avenues in biomedical research. RNA interference (RNAi) is now an important tool able to specifically inhibit the expression of almost any gene. The understanding of the molecular determinants of endocrine diseases has benefited a great deal from the new opportunities offered by the use of RNAi. Because RNAi is able to specifically inhibit the expression of particular genes it has great therapeutic potential, and the first clinical trials have already started. The delivery of RNAi in vivo, however, requires different methods to those used in vitro. RNAi uses several components of a cellular pathway devoted to the production of miRNAs, a class of naturally occurring small, noncoding RNA molecules that function as translational repressors. There is growing evidence that miRNAs play key regulatory roles in several cellular processes, such as proliferation, differentiation and apoptosis, and recent publications have demonstrated that alterations in miRNA function might be involved in endocrine diseases, including diabetes mellitus, and in endocrine cancer.


Assuntos
Doenças do Sistema Endócrino/terapia , Interferência de RNA , RNA não Traduzido/genética , Animais , Doenças do Sistema Endócrino/genética , Doenças do Sistema Endócrino/patologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA não Traduzido/metabolismo , Tecnologia Farmacêutica/métodos , Tecnologia Farmacêutica/tendências
3.
Mol Ther Nucleic Acids ; 3: e148, 2014 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-24549299

RESUMO

Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD) to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides) will be required. To investigate the dynamics and persistence of antisense 2'-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours-24 weeks). Oligonucleotide half-life was longer in heart (~65 days) compared with that in skeletal muscle, liver, and kidney (~35 days). Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days). Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3-8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2'-O-methyl phosphorothioate oligos used for the treatment of DMD.Molecular Therapy-Nucleic Acids (2014) 3, e148; doi:10.1038/mtna.2014.1; published online 18 February 2014.

4.
Nucleic Acid Ther ; 23(3): 228-37, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23634945

RESUMO

Antisense-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy. It aims to restore the dystrophin open reading frame by skipping exons with antisense oligonucleotides (AONs) to allow production of partly functional proteins. The approach is currently tested in phase 3 clinical trials, but dosing and maintenance regimens have not yet been well studied. This study compared pharmacokinetic and pharmacodynamic effects of different 2'-O-methyl phosphorothioate RNA AON dosing and maintenance regimens in the preclinical mdx mouse model. When comparing different dosing regimens over a period of 8 weeks, higher levels of AON, exon skipping, and protein were observed in muscle after low daily doses compared with large weekly doses. Secondly, after receiving a high loading dose (1,250 mg/kg) in the first week, mice treated with maintenance injections twice weekly for 8 weeks showed higher preservation of therapeutic effects than mice receiving less or no maintenance injections. In both cases, the regimen resulting in the highest AON and exon skipping levels in muscle also resulted in high AON levels in liver and kidneys. These studies underline the importance of balancing optimal AON efficacy and tolerable levels in non-target organs, which may be fine-tuned by further optimization of AON treatment regimens.


Assuntos
Distrofina/genética , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Fosforotioatos/farmacologia , Animais , Creatina Quinase/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Esquema de Medicação , Cálculos da Dosagem de Medicamento , Distrofina/agonistas , Distrofina/metabolismo , Éxons , Terapia Genética , Humanos , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Fosforotioatos/síntese química , Oligonucleotídeos Fosforotioatos/farmacocinética
5.
Hum Gene Ther ; 23(3): 262-73, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22017442

RESUMO

In Duchenne muscular dystrophy (DMD), dystrophin deficiency leading to progressive muscular degeneration is caused by frame-shifting mutations in the DMD gene. Antisense oligonucleotides (AONs) aim to restore the reading frame by skipping of a specific exon(s), thereby allowing the production of a shorter, but semifunctional protein, as is found in the mostly more mildly affected patients with Becker muscular dystrophy. AONs are currently being investigated in phase 3 placebo-controlled clinical trials. Most of the participating patients are treated symptomatically with corticosteroids (mainly predniso[lo]ne) to stabilize the muscle fibers, which might affect the uptake and/or efficiency of AONs. Therefore the effect of prednisolone on 2'-O-methyl phosphorothioate AON efficacy in patient-derived cultured muscle cells and the mdx mouse model (after local and systemic AON treatment) was assessed in this study. Both in vitro and in vivo skip efficiency and biomarker expression were comparable between saline- and prednisolone-cotreated cells and mice. After systemic exon 23-specific AON (23AON) treatment for 8 weeks, dystrophin was detectable in all treated mice. Western blot analyses indicated slightly higher dystrophin levels in prednisolone-treated mice, which might be explained by better muscle condition and consequently more target dystrophin pre-mRNA. In addition, fibrotic and regeneration biomarkers were normalized to some extent in prednisolone- and/or 23AON-treated mice. Overall these results show that the use of prednisone forms no barrier to participation in clinical trials with AONs.


Assuntos
Éxons , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/metabolismo , Prednisolona/administração & dosagem , Animais , Células Cultivadas , Distrofina/genética , Distrofina/metabolismo , Terapia Genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Prednisolona/uso terapêutico
6.
Genes Dev ; 21(5): 562-77, 2007 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-17344417

RESUMO

Little is known about the regulation and function of the Notch1 gene in negative control of human tumors. Here we show that Notch1 gene expression and activity are substantially down-modulated in keratinocyte cancer cell lines and tumors, with expression of this gene being under p53 control in these cells. Genetic suppression of Notch signaling in primary human keratinocytes is sufficient, together with activated ras, to cause aggressive squamous cell carcinoma formation. Similar tumor-promoting effects are also caused by in vivo treatment of mice, grafted with keratinocytes expressing oncogenic ras alone, with a pharmacological inhibitor of endogenous Notch signaling. These effects are linked with a lesser commitment of keratinocytes to differentiation, an expansion of stem cell populations, and a mechanism involving up-regulation of ROCK1/2 and MRCKalpha kinases, two key effectors of small Rho GTPases previously implicated in neoplastic progression. Thus, the Notch1 gene is a p53 target with a role in human tumor suppression through negative regulation of Rho effectors.


Assuntos
Carcinoma de Células Escamosas/genética , Genes Supressores de Tumor , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratinócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor Notch1/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Queratinócitos/citologia , Camundongos , Miotonina Proteína Quinase , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , RNA Interferente Pequeno , Receptor Notch1/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células-Tronco/citologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Quinases Associadas a rho
7.
Cell ; 121(6): 849-58, 2005 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-15960973

RESUMO

Activating mutations of RAS frequently occur in subsets of human cancers, indicating that RAS activation is important for tumorigenesis. However, a large proportion of these cancers still retain wild-type RAS alleles, suggesting that either the RAS pathway is activated in a distinct manner or another pathway is deregulated. To uncover novel tumor-suppressor genes, we screened an RNA-interference library for knockdown constructs that transform human primary cells in the absence of ectopically introduced oncogenic RAS. Here we report the identification of PITX1, whose inhibition induces the RAS pathway and tumorigenicity. Interestingly, we observed low expression of PITX1 in prostate and bladder tumors and in colon cancer cell lines containing wild-type RAS. Restoration of PITX1 in the colon cancer cells inhibited tumorigenicity in a wild-type RAS-dependent manner. Finally, we identified RASAL1, a RAS-GTPase-activating protein, as a transcription target through which PITX1 affects RAS function. Thus, PITX1 suppresses tumorigenicity by downregulating the RAS pathway through RASAL1.


Assuntos
Proteínas Ativadoras de GTPase/fisiologia , Genes Supressores de Tumor/fisiologia , Testes Genéticos/métodos , Proteínas de Homeodomínio/farmacologia , Fatores de Transcrição/farmacologia , Proteínas ras , Animais , Células CACO-2 , Linhagem Celular Tumoral , Proteínas Ativadoras de GTPase/efeitos dos fármacos , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Biblioteca Gênica , Vetores Genéticos/farmacologia , Células HeLa , Proteínas de Homeodomínio/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Nus , Fatores de Transcrição Box Pareados , Fenótipo , Interferência de RNA/fisiologia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Proteínas ras/antagonistas & inibidores , Proteínas ras/genética , Proteínas ras/fisiologia
8.
Blood ; 105(3): 1153-61, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15454490

RESUMO

Tumors have several mechanisms to escape from the immune system. One of these involves expression of intracellular anticytotoxic proteins that modulate the execution of cell death. Previously, we have shown that the serine protease inhibitor (serpin) SPI-6, which inactivates the cytotoxic protease granzyme B (GrB), is capable of preventing cytotoxic T lymphocyte (CTL)-mediated apoptosis. Despite its potent antiapoptotic activity, SPI-6 does not prevent membranolysis induced by cytotoxic lymphocytes. We now provide evidence that several colon carcinoma cell lines do resist membranolysis and that this protection is dependent on SPI-6 but also requires expression of a closely related serpin called SPI-CI (serine protease inhibitor involved in cytotoxicity inhibition). Expression of SPI-CI is absent from normal colon but observed in placenta, testis, early during embryogenesis, and in cytotoxic lymphocytes. SPI-CI encodes a chymotrypsin-specific inhibitor and irreversibly interacts with purified granzyme M. Moreover, SPI-CI can protect cells from purified perforin/GrM-induced lysis. Our data therefore indicate that SPI-CI is a novel immune escape molecule that acts in concert with SPI-6 to prevent cytotoxic lymphocyte-mediated killing of tumor cells.


Assuntos
Citotoxicidade Imunológica/imunologia , Proteínas de Membrana/imunologia , Serina Endopeptidases/imunologia , Serpinas/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Sequência de Bases , Primers do DNA , Endopeptidases/deficiência , Endopeptidases/genética , Endopeptidases/imunologia , Granzimas , Células Matadoras Ativadas por Linfocina/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Serpinas/genética , Baço/imunologia
9.
EMBO J ; 23(23): 4627-38, 2004 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-15538385

RESUMO

Genetic studies in Caenorhabditis elegans identified lin-9 to function together with the retinoblastoma homologue lin-35 in vulva differentiation. We have now identified a human homologue of Lin-9 (hLin-9) and provide evidence about its function in the mammalian pRB pathway. hLin-9 binds to pRB and cooperates with pRB in flat cell formation in Saos-2 cells. In addition, hLin-9 synergized with pRB and Cbfal to transactivate an osteoblast-specific reporter gene. In contrast, hLin-9 was not involved in pRB-mediated inhibition of cell cycle progression or repression of E2F-dependent transactivation. Consistent with these data, hLin-9 was able to associate with partially penetrant pRB mutants that do not bind to E2F, but retain the ability to activate transcription and to promote differentiation. hLin-9 can also inhibit oncogenic transformation, dependent on the presence of a functional pRB protein. RNAi-mediated knockdown of Lin-9 can substitute for the loss of pRB in transformation of human primary fibroblasts. These data suggest that hLin-9 has tumor-suppressing activities and that the ability of hLin-9 to inhibit transformation is mediated through its association with pRB.


Assuntos
Transformação Celular Neoplásica/metabolismo , Proteína do Retinoblastoma/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Forma Celular/genética , Forma Celular/fisiologia , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Ligação Proteica , Proteína do Retinoblastoma/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , Transcrição Gênica/fisiologia , Proteínas Supressoras de Tumor/genética
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