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1.
Nucleic Acids Res ; 50(22): 12657-12673, 2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36511872

RESUMO

Friedreich's ataxia is an incurable disease caused by frataxin (FXN) protein deficiency, which is mostly induced by GAA repeat expansion in intron 1 of the FXN gene. Here, we identified antisense oligonucleotides (ASOs), complementary to two regions within the first intron of FXN pre-mRNA, which could increase FXN mRNA by ∼2-fold in patient fibroblasts. The increase in FXN mRNA was confirmed by the identification of multiple overlapping FXN-activating ASOs at each region, two independent RNA quantification assays, and normalization by multiple housekeeping genes. Experiments on cells with the ASO-binding sites deleted indicate that the ASO-induced FXN activation was driven by indirect effects. RNA sequencing analyses showed that the two ASOs induced similar transcriptome-wide changes, which did not resemble the transcriptome of wild-type cells. This RNA-seq analysis did not identify directly base-paired off-target genes shared across ASOs. Mismatch studies identified two guanosine-rich motifs (CCGG and G4) within the ASOs that were required for FXN activation. The phosphorodiamidate morpholino oligomer analogs of our ASOs did not activate FXN, pointing to a PS-backbone-mediated effect. Our study demonstrates the importance of multiple, detailed control experiments and target validation in oligonucleotide studies employing novel mechanisms such as gene activation.


Assuntos
Ataxia de Friedreich , Regulação da Expressão Gênica , Oligonucleotídeos Antissenso , Humanos , Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/metabolismo , RNA Mensageiro/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Frataxina
2.
Cold Spring Harb Protoc ; 2019(10)2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575796

RESUMO

RNA samples prepared using monophasic lysis reagents may contain small amounts of contaminating genomic DNA, which must be removed if the RNA will be used in subsequent analyses such as reverse transcriptase-polymerase chain reaction (RT-PCR) or quantitative real-time RT-PCR. In addition, the presence of contaminating DNA can render the quantitative determination of RNA in a sample inaccurate. The most common and effective method for removing trace to moderate amounts of DNA contamination from RNA samples is digestion with DNase I, as described here.


Assuntos
Contaminação por DNA , DNA/genética , Desoxirribonucleases/metabolismo , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ribonucleases/metabolismo , DNA/isolamento & purificação , DNA/metabolismo , Desoxirribonuclease I/antagonistas & inibidores , Desoxirribonuclease I/metabolismo , Ácido Edético/química , Ácido Edético/metabolismo , Ácido Edético/farmacologia , Ativação Enzimática/efeitos dos fármacos , Hidrólise , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
3.
Int J Radiat Oncol Biol Phys ; 85(2): 321-8, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22687197

RESUMO

PURPOSE: Despite recent advances in the management of high-grade and recurrent gliomas, survival remains poor. Antiangiogenic therapy has been shown to be efficacious in the treatment of high-grade gliomas both in preclinical models and in clinical trials. We sought to determine the safety and maximum tolerated dose of sorafenib when combined with both radiation and temozolomide in the primary setting or radiation alone in the recurrent setting. METHODS AND MATERIALS: This was a preclinical study and an open-label phase I dose escalation trial. Multiple glioma cell lines were analyzed for viability after treatment with radiation, temozolomide, or sorafenib or combinations of them. For patients with primary disease, sorafenib was given concurrently with temozolomide (75 mg/m(2)) and 60 Gy radiation, for 30 days after completion of radiation. For patients with recurrent disease, sorafenib was combined with a hypofractionated course of radiation (35 Gy in 10 fractions). RESULTS: Cell viability was significantly reduced with the combination of radiation, temozolomide, and sorafenib or radiation and sorafenib. Eighteen patients (11 in the primary cohort, 7 in the recurrent cohort) were enrolled onto this trial approved by the institutional review board. All patients completed the planned course of radiation therapy. The most common toxicities were hematologic, fatigue, and rash. There were 18 grade 3 or higher toxicities. The median overall survival was 18 months for the entire population. CONCLUSIONS: Sorafenib can be safely combined with radiation and temozolomide in patients with high-grade glioma and with radiation alone in patients with recurrent glioma. The recommended phase II dose of sorafenib is 200 mg twice daily when combined with temozolomide and radiation and 400 mg with radiation alone. To our knowledge, this is the first publication of concurrent sorafenib with radiation monotherapy or combined with radiation and temozolomide.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/terapia , Quimiorradioterapia/métodos , Dacarbazina/análogos & derivados , Glioma/terapia , Recidiva Local de Neoplasia/terapia , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimiorradioterapia/efeitos adversos , Dacarbazina/administração & dosagem , Dacarbazina/uso terapêutico , Feminino , Glioma/irrigação sanguínea , Glioma/mortalidade , Glioma/patologia , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Proteínas de Neoplasias/sangue , Recidiva Local de Neoplasia/irrigação sanguínea , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Niacinamida/administração & dosagem , Niacinamida/efeitos adversos , Niacinamida/uso terapêutico , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/efeitos adversos , Dosagem Radioterapêutica , Sorafenibe , Temozolomida , Fator A de Crescimento do Endotélio Vascular/sangue
4.
Proc Natl Acad Sci U S A ; 108(7): 2873-8, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21282644

RESUMO

We utilized gene expression profiling of a comprehensive panel of purified developmentally defined normal murine B cells to identify unique transcriptional signatures for each subset. To elucidate transcription factor activities that function in a stage-specific fashion, we used gene sets that share transcription factor targets and found that germinal center B cells had a robust enrichment of up-regulated and down-regulated signatures compared with the other B-cell subsets. Notably, we found Yy1 and its targets to be central regulators of the germinal center B (GCB)-specific transcriptional program with binding of Yy1 to select signature genes in GCB cells, and translation of the Yy1 signatures to human GCB cells. We then tested whether our newly generated, stage-specific transcriptional signatures could be used to link murine lymphoma models to stages of normal B-cell development. Although each of the molecularly defined murine lymphoma models conserved certain stage-specific features of normal B-cell development, there was a significant alteration of the normal differentiation signature following malignant transformation. These findings offer important tools and insights for elucidating differences between normal and malignant B cells.


Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Regulação da Expressão Gênica/imunologia , Centro Germinativo/metabolismo , Linfoma/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Subpopulações de Linfócitos B/citologia , Diferenciação Celular/imunologia , Imunoprecipitação da Cromatina , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Camundongos
5.
Nat Med ; 16(6): 671-7, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20495567

RESUMO

Activating transcription factor-5 (ATF5) is highly expressed in malignant glioma and has a key role in promoting cell survival. Here we perform a genome-wide RNAi screen to identify transcriptional regulators of ATF5. Our results reveal an essential survival pathway in malignant glioma, whereby activation of a RAS-mitogen-activated protein kinase or phosphoinositide-3-kinase signaling cascade leads to induction of the transcription factor cAMP response element-binding protein-3-like-2 (CREB3L2), which directly activates ATF5 expression. ATF5, in turn, promotes survival by stimulating transcription of myeloid cell leukemia sequence-1 (MCL1), an antiapoptotic B cell leukemia-2 family member. Analysis of human malignant glioma samples indicates that ATF5 expression inversely correlates with disease prognosis. The RAF kinase inhibitor sorafenib suppresses ATF5 expression in glioma stem cells and inhibits malignant glioma growth in cell culture and mouse models. Our results demonstrate that ATF5 is essential in malignant glioma genesis and reveal that the ATF5-mediated survival pathway described here provides potential therapeutic targets for treatment of malignant glioma.


Assuntos
Fatores Ativadores da Transcrição/genética , Neoplasias Encefálicas/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Perfilação da Expressão Gênica , Glioma/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Fatores Ativadores da Transcrição/antagonistas & inibidores , Fatores Ativadores da Transcrição/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Benzenossulfonatos/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Sequência 1 de Leucemia de Células Mieloides , Transplante de Neoplasias , Niacinamida/análogos & derivados , Compostos de Fenilureia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Piridinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Sorafenibe , Células Tumorais Cultivadas
6.
Mol Cell Biol ; 23(13): 4532-41, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12808095

RESUMO

Apoptosis of mature T lymphocytes preserves immune system homeostasis by counteracting transient increases in T-cell number. This process is regulated, at least in part, by the cytokine interleukin 2 (IL-2): T cells deprived of IL-2 undergo apoptosis. The mechanism of apoptosis induction by IL-2 deprivation remains to be determined but is known to require RNA synthesis, implying the existence of transcriptionally activated genes whose products induce cell death. To identify such genes, we have performed expression profiling in IL-2-dependent T cells following cytokine deprivation. Our results reveal an intricate transcriptional program entailing the induction of known proapoptotic factors and the simultaneous repression of known antiapoptotic factors. Surprisingly, one gene whose transcription substantially increased was RC3 (also called neurogranin), which encodes a calmodulin binding protein thought to be a neural-specific factor involved in learning and memory. We show that ectopic expression of RC3 in IL-2-dependent T cells increases the intracellular Ca(2+) concentration and induces apoptosis even in the presence of cytokine. Buffering the Ca(2+) increase with the cytoplasmic Ca(2+) chelator BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N1,N-tetraacetic acid] blocks RC3-induced apoptosis, indicating that the rise in intracellular Ca(2+) is required for apoptotic death. RC3 mutants unable to bind calmodulin fail to increase intracellular Ca(2+) levels and to induce apoptosis. Based upon these results, we propose that IL-2 deprivation raises the level of RC3 and other apoptotic factors, which induce apoptosis by increasing the intracellular Ca(2+) concentration.


Assuntos
Apoptose , Proteínas de Ligação a Calmodulina/fisiologia , Ácido Egtázico/análogos & derivados , Interleucina-2/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Transcrição Gênica , Cálcio/metabolismo , Morte Celular , DNA Complementar/metabolismo , Ácido Egtázico/farmacologia , Citometria de Fluxo , Vetores Genéticos , Humanos , Células Jurkat , Neurogranina , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , RNA/metabolismo , Espectrometria de Fluorescência , Fatores de Tempo , Ativação Transcricional , Transfecção
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