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1.
Genes Dev ; 30(19): 2152-2157, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27798842

RESUMO

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.


Assuntos
Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Mutações Sintéticas Letais/genética , Trissacarídeos/genética , Animais , Apoptose/genética , Proteínas de Ligação a DNA/metabolismo , Epistasia Genética , Instabilidade Genômica/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Tolerância a Radiação/genética , Trissacarídeos/metabolismo
2.
J Fluoresc ; 2023 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-37736833

RESUMO

Cellular therapy development and manufacturing has focused on providing novel therapeutic cell-based products for various diseases. The International Organization for Standardization (ISO) has provided guidance on critical quality attributes (CQAs) that shall be considered when testing and releasing cellular therapeutic products. Cell count and viability measurements are two of the CQAs that are determined during development, manufacturing, testing, and product release. The ISO Cell Counting Standard Part 1 and 2 addressed the needs for improving the quality of cell counting results. However, there is currently no guidance on the qualification and selection of a fit-for-purpose cell viability detection method. In this work, we present strategies for the characterization and comparison of AO/PI and AO/DAPI staining methods using the heat-killed (HK) and low temperature/nutrient-deprived (LT/ND) cell death models to evaluate the comparability of cell viability measurements and identify potential causes of differences. We compared the AO/PI and AO/DAPI staining methods using HK and LT/ND-generated dead cells, investigated the staining time effects on cell viability measurements, and determined their viability linearity with different mixtures of live and dead cells. Furthermore, we validated AO/PI and AO/DAPI cell viability measurement with a long-term cell proliferation assay. Finally, we demonstrate a practical example of cell viability measurement comparison using AO/PI and AO/DAPI on antibiotic-selected transduced Jurkat and THP-1 cells to select a fit-for-purpose method for functional genomics screening. The proposed strategies may potentially enable scientists to properly characterize, compare, and select cell viability detection methods that are critical for cellular therapeutic product development and manufacturing.

3.
Genes Dev ; 23(11): 1327-37, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19487573

RESUMO

Activated oncogenic signaling is central to the development of nearly all forms of cancer, including the most common class of primary brain tumor, glioma. Research over the last two decades has revealed the particular importance of the Akt pathway, and its molecular antagonist PTEN (phosphatase and tensin homolog), in the process of gliomagenesis. Recent studies have also demonstrated that microRNAs (miRNAs) may be responsible for the modulation of cancer-implicated genes in tumors. Here we report the identification miR-26a as a direct regulator of PTEN expression. We also show that miR-26a is frequently amplified at the DNA level in human glioma, most often in association with monoallelic PTEN loss. Finally, we demonstrate that miR-26a-mediated PTEN repression in a murine glioma model both enhances de novo tumor formation and precludes loss of heterozygosity and the PTEN locus. Our results document a new epigenetic mechanism for PTEN regulation in glioma and further highlight dysregulation of Akt signaling as crucial to the development of these tumors.


Assuntos
Regulação Neoplásica da Expressão Gênica , Glioma/fisiopatologia , PTEN Fosfo-Hidrolase/metabolismo , Animais , Células Cultivadas , DNA Helicases/metabolismo , Modelos Animais de Doenças , Estimativa de Kaplan-Meier , Perda de Heterozigosidade , Camundongos , MicroRNAs/metabolismo , Células NIH 3T3 , PTEN Fosfo-Hidrolase/genética
4.
Circulation ; 123(3): 282-91, 2011 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-21220732

RESUMO

BACKGROUND: Diabetes mellitus impairs endothelial cell (EC) function and postischemic reparative neovascularization by molecular mechanisms that are not fully understood. microRNAs negatively regulate the expression of target genes mainly by interaction in their 3' untranslated region. METHODS AND RESULTS: We found that microRNA-503 (miR-503) expression in ECs is upregulated in culture conditions mimicking diabetes mellitus (high D-glucose) and ischemia-associated starvation (low growth factors). Under normal culture conditions, lentivirus-mediated miR-503-forced expression inhibited EC proliferation, migration, and network formation on Matrigel (comparisons versus lentivirus.GFP control). Conversely, blocking miR-503 activity by either adenovirus-mediated transfer of a miR-503 decoy (Ad.decoymiR-503) or by antimiR-503 (antisense oligonucleotide) improved the functional capacities of ECs cultured under high D-glucose/low growth factors. We identified CCNE1 and cdc25A as direct miR-503 targets which are downregulated by high glucose/low growth factors in ECs. Next, we obtained evidence that miR-503 expression is increased in ischemic limb muscles of streptozotocin-diabetic mice and in ECs enriched from these muscles. Moreover, Ad.decoymiR-503 delivery to the ischemic adductor of diabetic mice corrected diabetes mellitus-induced impairment of postischemic angiogenesis and blood flow recovery. We finally investigated miR-503 and target gene expression in muscular specimens from the amputated ischemic legs of diabetic patients. As controls, calf biopsies of nondiabetic and nonischemic patients undergoing saphenous vein stripping were used. In diabetic muscles, miR-503 expression was remarkably higher, and it inversely correlated with cdc25 protein expression. Plasma miR-503 levels were also elevated in the diabetic individuals. CONCLUSIONS: Our data suggest miR-503 as a possible therapeutic target in diabetic patients with critical limb ischemia.


Assuntos
Diabetes Mellitus Experimental , Isquemia , MicroRNAs/fisiologia , Neovascularização Fisiológica/fisiologia , Animais , Biópsia , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Extremidades/irrigação sanguínea , Expressão Gênica/fisiologia , Glucose/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Isquemia/genética , Isquemia/patologia , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos , Músculo Esquelético/patologia , Músculo Esquelético/fisiologia , Veias Umbilicais/citologia , Fosfatases cdc25/genética
5.
Sci Transl Med ; 13(615): eabh1486, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34644148

RESUMO

Discovery of small-molecule degraders that activate ubiquitin ligase­mediated ubiquitination and degradation of targeted oncoproteins in cancer cells has been an elusive therapeutic strategy. Here, we report a cancer cell­based drug screen of the NCI drug-like compounds library that enabled identification of small-molecule degraders of the small ubiquitin-related modifier 1 (SUMO1). Structure-activity relationship studies of analogs of the hit compound CPD1 led to identification of a lead compound HB007 with improved properties and anticancer potency in vitro and in vivo. A genome-scale CRISPR-Cas9 knockout screen identified the substrate receptor F-box protein 42 (FBXO42) of cullin 1 (CUL1) E3 ubiquitin ligase as required for HB007 activity. Using HB007 pull-down proteomics assays, we pinpointed HB007's binding protein as the cytoplasmic activation/proliferation-associated protein 1 (CAPRIN1). Biolayer interferometry and compound competitive immunoblot assays confirmed the selectivity of HB007's binding to CAPRIN1. When bound to CAPRIN1, HB007 induced the interaction of CAPRIN1 with FBXO42. FBXO42 then recruited SUMO1 to the CAPRIN1-CUL1-FBXO42 ubiquitin ligase complex, where SUMO1 was ubiquitinated in several of human cancer cells. HB007 selectively degraded SUMO1 in patient tumor­derived xenografts implanted into mice. Systemic administration of HB007 inhibited the progression of patient-derived brain, breast, colon, and lung cancers in mice and increased survival of the animals. This cancer cell­based screening approach enabled discovery of a small-molecule degrader of SUMO1 and may be useful for identifying other small-molecule degraders of oncoproteins.


Assuntos
Neoplasias , Proteína SUMO-1 , Animais , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Ubiquitinação
6.
Mol Cancer ; 9: 167, 2010 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-20579385

RESUMO

BACKGROUND: A substantial number of microRNAs (miRNAs) is subject to epigenetic silencing in cancer. Although epigenetic silencing of tumour suppressor genes is an important feature of cervical cancer, little is known about epigenetic silencing of miRNAs. Since DNA methylation-based silencing of hsa-miR-124 occurs in various human cancers, we studied the frequency and functional effects of hsa-miR-124 methylation in cervical carcinogenesis. RESULTS: Quantitative MSP analysis of all 3 loci encoding the mature hsa-miR-124 (hsa-miR-124-1/-2/-3) showed methylation in cervical cancer cell lines SiHa, CaSki and HeLa as well as in late passages of human papillomavirus (HPV) type 16 or 18 immortalised keratinocytes. Treatment of SiHa cells with a demethylating agent reduced hsa-miR-124 methylation levels and induced hsa-miR-124 expression. In HPV-immortalised keratinocytes increased methylation levels were related to reduced hsa-miR-124 expression and higher mRNA expression of IGFBP7, a potential hsa-miR-124 target gene. Ectopic hsa-miR-124 expression in SiHa and CaSki cells decreased proliferation rates and migratory capacity. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 139 cervical tissue specimens showed an increasing methylation frequency from 0% in normal tissues up to 93% in cervical carcinomas. Increased methylation levels of hsa-miR-124-1 and hsa-miR-124-2 were significantly correlated with reduced hsa-miR-124 expression in cervical tissue specimens. Combined hsa-miR-124-1 and/or hsa-miR-124-2 methylation analysis of 43 cervical scrapes of high-risk HPV positive women was predictive of underlying high-grade lesions. CONCLUSIONS: DNA methylation-based silencing of hsa-miR-124 is functionally involved in cervical carcinogenesis and may provide a valuable marker for improved detection of cervical cancer and its high-grade precursor lesions.


Assuntos
Metilação de DNA , Inativação Gênica , MicroRNAs/fisiologia , Neoplasias do Colo do Útero/genética , Adulto , Idoso , Alphapapillomavirus/fisiologia , Sequência de Bases , Primers do DNA , Feminino , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , MicroRNAs/genética , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
RNA ; 14(5): 872-7, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18367714

RESUMO

MicroRNAs (miRNAs) are small noncoding RNA molecules of 20-24 nucleotides that regulate gene expression. In animals, miRNAs form imperfect interactions with sequences in the 3' Untranslated region (3'UTR) of mRNAs, causing translational inhibition and mRNA decay. In contrast, plant miRNAs mostly associate with protein coding regions. Here we show that human miR-148 represses DNA methyltransferase 3b (Dnmt3b) gene expression through a region in its coding sequence. This region is evolutionary conserved and present in the Dnmt3b splice variants Dnmt3b1, Dnmt3b2, and Dnmt3b4, but not in the abundantly expressed Dnmt3b3. Whereas overexpression of miR-148 results in decreased DNMT3b1 expression, short-hairpin RNA-mediated miR-148 repression leads to an increase in DNMT3b1 expression. Interestingly, mutating the putative miR-148 target site in Dnmt3b1 abolishes regulation by miR-148. Moreover, endogenous Dnmt3b3 mRNA, which lacks the putative miR-148 target site, is resistant to miR-148-mediated regulation. Thus, our results demonstrate that the coding sequence of Dnmt3b mediates regulation by the miR-148 family. More generally, we provide evidence that coding regions of human genes can be targeted by miRNAs, and that such a mechanism might play a role in determining the relative abundance of different splice variants.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , MicroRNAs/genética , Processamento Alternativo , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido Nucleico , Transfecção , DNA Metiltransferase 3B
8.
SLAS Discov ; 25(3): 233-240, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31658850

RESUMO

The discovery of CRISPR-Cas9 systems has fueled a rapid expansion of gene editing adoption and has impacted pharmaceutical and biotechnology research substantially. Here, gene editing is used at an industrial scale to identify and validate new biological targets for precision medicines, with functional genomic screening having an increasingly important role. Functional genomic strategies provide a crucial link between observed biological phenomena and the genes that influence and drive those phenomena. Although such studies are not new, the use of CRISPR-Cas9 systems in this arena is providing more robust datasets for target identification and validation. CRISPR-based screening approaches are also useful later in the drug development pipeline for understanding drug resistance and sensitivity ahead of entering clinical trials. This review examines the developing landscape for CRISPR screening technologies within the pharmaceutical industry and explores the next steps for this constantly evolving screening platform.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Humanos
9.
CRISPR J ; 3(3): 211-222, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-33054419

RESUMO

Functional genomic screening with CRISPR has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. However, some experimental paradigms prove especially challenging and require carefully and appropriately adapted screening approaches. In particular, negative selection (or sensitivity) screening, often the most experimentally desirable modality of screening, has remained a challenge in drug discovery. Here we assess whether our new, modular genome-wide pooled CRISPR library can improve negative selection CRISPR screening and add utility throughout the drug development pipeline. Our pooled library is split into three parts, allowing it to be scaled to accommodate the experimental challenges encountered during drug development, such as target identification using unlimited cell numbers compared with target identification studies for cell populations where cell numbers are limiting. To test our new library, we chose to look for drug-gene interactions using a well-described small molecule inhibitor targeting poly(ADP-ribose) polymerase 1 (PARP1), and in particular to identify genes which sensitise cells to this drug. We simulate hit identification and performance using each library partition and support these findings through orthogonal drug combination cell panel screening. We also compare our data with a recently published CRISPR sensitivity dataset obtained using the same PARP1 inhibitor. Overall, our data indicate that generating a comprehensive CRISPR knockout screening library where the number of guides can be scaled to suit the biological question being addressed allows a library to have multiple uses throughout the drug development pipeline, and that initial validation of hits can be achieved through high-throughput cell panels screens where clinical grade chemical or biological matter exist.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desenvolvimento de Medicamentos , Biblioteca Gênica , Proteínas de Ligação a DNA , Técnicas de Inativação de Genes , Células HT29 , Ensaios de Triagem em Larga Escala , Humanos , Preparações Farmacêuticas , RNA Guia de Cinetoplastídeos/genética
10.
Adv Exp Med Biol ; 604: 17-46, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17695719

RESUMO

Endogenous small RNAs (miRNAs) regulate gene expression by mechanisms conserved across metazoans. While the number of verified human miRNAs is still expanding, only few have been functionally annotated. To perform genetic screens for novel functions of miRNAs, we developed a library of vectors expressing the majority of cloned human miRNAs and created corresponding DNA barcode arrays. In a screen for miRNAs that cooperate with oncogenes in cellular transformation, we identified miR-372 and miR-373, each permitting proliferation and tumorigenesis of primary human cells that harbor both oncogenic RAS and active wild-type p53. These miRNAs neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumorsuppressor LATS2. We provide evidence that these miRNAs are potential novel oncogenes participating in the development of human testicular germ cell tumors by numbing the p53 pathway, thus allowing tumorigenic growth in the presence of wild-type p53.


Assuntos
Regulação da Expressão Gênica , Técnicas Genéticas , MicroRNAs/genética , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Testiculares/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Testes Genéticos/métodos , Humanos , Masculino , Camundongos , Camundongos Nus , MicroRNAs/química , Dados de Sequência Molecular , Transplante de Neoplasias , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas ras/metabolismo
11.
Sci Rep ; 7(1): 17693, 2017 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-29255251

RESUMO

Pooled CRISPR-Cas9 knock out screens provide a valuable addition to the methods available for novel drug target identification and validation. However, where gene editing is targeted to amplified loci, the resulting multiple DNA cleavage events can be a cause of false positive hit identification. The generation of nuclease deficient versions of Cas9 has enabled the development of two additional techniques - CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) - that enable the repression or overexpression, respectively, of target genes. Here we report the first direct combination of all three approaches (CRISPRko, CRISPRi and CRISPRa) in the context of genome-wide screens to identify components that influence resistance and sensitivity to the BRAF inhibitor, vemurafenib. The pairing of both loss- and gain-of-function datasets reveals complex gene networks which control drug response and illustrates how such data can add substantial confidence to target identification and validation analyses.


Assuntos
Resistência a Medicamentos/genética , Técnicas de Inativação de Genes/métodos , Redes Reguladoras de Genes/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Clivagem do DNA , Avaliação Pré-Clínica de Medicamentos/métodos , Endonucleases/genética , Edição de Genes/métodos , Regulação da Expressão Gênica/genética , Genoma/genética , Humanos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Vemurafenib/farmacologia
12.
Sci Rep ; 6: 24356, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27079678

RESUMO

The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system's specificity. Recently a "double-nicking" strategy using catalytic mutant Cas9(D10A) nickase has been developed to minimise off-target effects. Here, we describe a Cas9(D10A)-based screening approach that combines an All-in-One Cas9(D10A) nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities.


Assuntos
Sistemas CRISPR-Cas , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Edição de Genes , Genótipo , Fenótipo , Alelos , Sequência de Bases , Linhagem Celular , Descoberta de Drogas , Técnicas de Inativação de Genes , Ordem dos Genes , Marcação de Genes , Loci Gênicos , Vetores Genéticos/genética , Genômica/métodos , Ensaios de Triagem em Larga Escala , Humanos , Mutagênese , RNA Guia de Cinetoplastídeos , Fator A de Crescimento do Endotélio Vascular/genética
13.
Cell Rep ; 11(5): 704-14, 2015 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-25921528

RESUMO

The activities of many DNA-repair proteins are controlled through reversible covalent modification by ubiquitin and ubiquitin-like molecules. Nonhomologous end-joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in mammalian cells and is initiated by DSB ends being recognized by the Ku70/Ku80 (Ku) heterodimer. By using MLN4924, an anti-cancer drug in clinical trials that specifically inhibits conjugation of the ubiquitin-like protein, NEDD8, to target proteins, we demonstrate that NEDD8 accumulation at DNA-damage sites is a highly dynamic process. In addition, we show that depleting cells of the NEDD8 E2-conjugating enzyme, UBE2M, yields ionizing radiation hypersensitivity and reduced cell survival following NHEJ. Finally, we demonstrate that neddylation promotes Ku ubiquitylation after DNA damage and release of Ku and Ku-associated proteins from damage sites following repair. These studies provide insights into how the NHEJ core complex dissociates from repair sites and highlight its importance for cell survival following DSB induction.


Assuntos
Antígenos Nucleares/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Ubiquitinas/metabolismo , Antígenos Nucleares/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ciclopentanos/toxicidade , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/química , Histonas/metabolismo , Humanos , Autoantígeno Ku , Proteína NEDD8 , Ligação Proteica , Estrutura Terciária de Proteína , Proteômica , Pirimidinas/toxicidade , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Radiação Ionizante , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinas/antagonistas & inibidores
14.
Nat Cell Biol ; 16(10): 1016-26, 1-8, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25194926

RESUMO

DNA double-strand breaks (DSBs) are perhaps the most toxic of all DNA lesions, with defects in the DNA-damage response to DSBs being associated with various human diseases. Although it is known that DSB repair pathways are tightly regulated by ubiquitylation, we do not yet have a comprehensive understanding of how deubiquitylating enzymes (DUBs) function in DSB responses. Here, by carrying out a multidimensional screening strategy for human DUBs, we identify several with hitherto unknown links to DSB repair, the G2/M DNA-damage checkpoint and genome-integrity maintenance. Phylogenetic analyses reveal functional clustering within certain DUB subgroups, suggesting evolutionally conserved functions and/or related modes of action. Furthermore, we establish that the DUB UCHL5 regulates DSB resection and repair by homologous recombination through protecting its interactor, NFRKB, from degradation. Collectively, our findings extend the list of DUBs promoting the maintenance of genome integrity, and highlight their potential as therapeutic targets for cancer.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Enzimas/metabolismo , Ubiquitinação , Linhagem Celular Tumoral , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Enzimas/classificação , Enzimas/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopia Confocal , Filogenia , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo
15.
Cancer Res ; 74(15): 4145-56, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24876105

RESUMO

Telomeres consist of DNA tandem repeats that recruit the multiprotein complex shelterin to build a chromatin structure that protects chromosome ends. Although cancer formation is linked to alterations in telomere homeostasis, there is little understanding of how shelterin function is limited in cancer cells. Using a small-scale screening approach, we identified miR-155 as a key regulator in breast cancer cell expression of the shelterin component TERF1 (TRF1). miR-155 targeted a conserved sequence motif in the 3'UTR of TRF1, resulting in its translational repression. miR-155 was upregulated commonly in breast cancer specimens, as associated with reduced TRF1 protein expression, metastasis-free survival, and relapse-free survival in estrogen receptor-positive cases. Modulating miR-155 expression in cells altered TRF1 levels and TRF1 abundance at telomeres. Compromising TRF1 expression by elevating miR-155 increased telomere fragility and altered the structure of metaphase chromosomes. In contrast, reducing miR-155 levels improved telomere function and genomic stability. These results implied that miR-155 upregulation antagonizes telomere integrity in breast cancer cells, increasing genomic instability linked to poor clinical outcome in estrogen receptor-positive disease. Our work argued that miRNA-dependent regulation of shelterin function has a clinically significant impact on telomere function, suggesting the existence of "telo-miRNAs" that have an impact on cancer and aging.


Assuntos
Neoplasias da Mama/genética , MicroRNAs/genética , Telômero/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Neoplasias da Mama/metabolismo , Técnicas de Cultura de Células , Feminino , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , MicroRNAs/metabolismo , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Telômero/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/metabolismo , Transfecção
16.
Autophagy ; 8(2): 165-76, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22248718

RESUMO

Macroautophagy (autophagy) is the major intracellular degradation pathway for long-lived proteins and organelles. It helps the cell to survive a spectrum of stressful conditions including starvation, growth factor deprivation and misfolded protein accumulation. Moreover, abnormalities of autophagy play a role in major health problems including cancer and neurodegenerative diseases. Yet, mechanisms controlling autophagic activity are not fully understood. Here, we describe hsa-miR-376b (miR-376b) as a new microRNA (miRNA) regulating autophagy. We showed that miR-376b expression attenuated starvation- and rapamycin-induced autophagy in MCF-7 and Huh-7 cells. We discovered autophagy proteins ATG4C and BECN1 (Beclin 1) as cellular targets of miR-376b. Indeed, upon miRNA overexpression, both mRNA and protein levels of ATG4C and BECN1 were decreased. miR-376b target sequences were present in the 3' UTR of ATG4C and BECN1 mRNAs and introduction of mutations abolished their miR-376b responsiveness. Antagomir-mediated inactivation of the endogenous miR-376b led to an increase in ATG4C and BECN1 levels. Therefore, miR-376b controls autophagy by directly regulating intracellular levels of two key autophagy proteins, ATG4C and BECN1.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Cisteína Endopeptidases/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia , Sequência de Bases , Proteína Beclina-1 , Linhagem Celular Tumoral , Cisteína Endopeptidases/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/genética , MicroRNAs/genética , Modelos Biológicos , Dados de Sequência Molecular , Oligorribonucleotídeos/farmacologia , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Proteína Sequestossoma-1 , Sirolimo/farmacologia , Inanição , Serina-Treonina Quinases TOR/metabolismo
17.
Nat Commun ; 2: 513, 2011 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-22027593

RESUMO

MicroRNAs (miRNAs) interact with 3'-untranslated regions of messenger RNAs to restrict expression of most protein-coding genes during normal development and cancer. RNA-binding proteins (RBPs) can control the biogenesis, stability and activity of miRNAs. Here we identify RBM38 in a genetic screen for RBPs whose expression controls miRNA access to target mRNAs. RBM38 is induced by p53 and its ability to modulate miRNA-mediated repression is required for proper p53 function. In contrast, RBM38 shows lower propensity to block the action of the p53-controlled miR-34a on SIRT1. Target selectivity is determined by the interaction of RBM38 with uridine-rich regions near miRNA target sequences. Furthermore, in large cohorts of human breast cancer, reduced RBM38 expression by promoter hypermethylation correlates with wild-type p53 status. Thus, our results indicate a novel layer of p53 gene regulation, which is required for its tumour suppressive function.


Assuntos
MicroRNAs/genética , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Regiões 3' não Traduzidas , Ciclo Celular , Linhagem Celular Tumoral , Metilação de DNA , Citometria de Fluxo , Humanos , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real
18.
Stem Cells Dev ; 19(6): 877-85, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19795981

RESUMO

Unrestricted somatic stem cells (USSCs) have been recently identified in human umbilical cord blood and have been shown to differentiate into lineages representing all 3 germ layers. To characterize microRNAs that may regulate osteogenic differentiation of USSCs, we carried out expression analysis for 157 microRNAs using quantitative RT-PCR before and after osteogenic induction (t = 0.5, 24, 72, 168, 216 h). Three microRNAs, hsa-miR-135b, hsa-miR-224, and hsa-miR-31, were consistently down-regulated during osteogenesis of USSC line 1. Hsa-miR-135b was shown to be the most profoundly down-regulated in osteogenesis of USSC line 1 and further confirmed to be down-regulated in the osteogenic differentiation of 2 additional USSC lines. Function of hsa-miR-135b in osteogenesis of USSCs was examined by retroviral overexpression, which resulted in an evident decreased mineralization, indicating that hsa-miR-135b down-regulation is functionally important for full osteogenic differentiation of USSCs. MicroRNAs have been shown to regulate negatively expression of their target gene(s). To identify putative targets of hsa-miR-135b, we performed cDNA microarray expression analysis. We selected in total 10 transcripts that were down-regulated (>or=2-fold) in response to hsa-miR-135b overexpression at day 7 and day 9 of osteogenic differentiation. The function of most of these targets in human osteogenesis is unknown and requires further investigation. Markedly, quantitative RT-PCR data showed decreased expression of osteogenic markers IBSP and Osterix, both known to be involved in bone mineralization, in osteogenesis of USSCs that overexpress hsa-miR-135b. This finding suggests that hsa-miR-135b may control osteoblastic differentiation of USSCs by regulating expression of bone-related genes.


Assuntos
Calcificação Fisiológica/genética , Diferenciação Celular/genética , MicroRNAs/metabolismo , Osteogênese/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética
19.
Cancer Res ; 68(14): 5795-802, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18632633

RESUMO

Inactivation of the adenomatous polyposis coli (APC) gene is a major initiating event in colorectal tumorigenesis. Most of the mutations in APC generate premature stop codons leading to truncated proteins that have lost beta-catenin binding sites. APC-free beta-catenin stimulates the Wnt signaling pathway, leading to active transcription of target genes. In the current study, we describe a novel mechanism for APC regulation. We show that miR-135a&b target the 3' untranslated region of APC, suppress its expression, and induce downstream Wnt pathway activity. Interestingly, we find a considerable up-regulation of miR-135a&b in colorectal adenomas and carcinomas, which significantly correlated with low APC mRNA levels. This genetic interaction is also preserved in full-blown cancer cell lines expressing miR-135a&b, regardless of the mutational status of APC. Thus, our results uncover a miRNA-mediated mechanism for the control of APC expression and Wnt pathway activity, and suggest its contribution to colorectal cancer pathogenesis.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Regiões 3' não Traduzidas , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Células HeLa , Humanos , Modelos Biológicos , Regiões Promotoras Genéticas , Transdução de Sinais , Proteínas Wnt/metabolismo
20.
Nat Cell Biol ; 10(2): 202-10, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18193036

RESUMO

MicroRNAs (miRNAs) are single-stranded, noncoding RNAs that are important in many biological processes. Although the oncogenic and tumour-suppressive functions of several miRNAs have been characterized, the role of miRNAs in mediating tumour metastasis was addressed only recently and still remains largely unexplored. To identify potential metastasis-promoting miRNAs, we set up a genetic screen using a non-metastatic, human breast tumour cell line that was transduced with a miRNA-expression library and subjected to a trans-well migration assay. We found that human miR-373 and miR-520c stimulated cancer cell migration and invasion in vitro and in vivo, and that certain cancer cell lines depend on endogenous miR-373 activity to migrate efficiently. Mechanistically, the migration phenotype of miR-373 and miR-520c can be explained by suppression of CD44. We found significant upregulation of miR-373 in clinical breast cancer metastasis samples that correlated inversely with CD44 expression. Taken together, our findings indicate that miRNAs are involved in tumour migration and invasion, and implicate miR-373 and miR-520c as metastasis-promoting miRNAs.


Assuntos
Movimento Celular/fisiologia , MicroRNAs/fisiologia , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ensaios de Migração Celular , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Metástase Linfática , Masculino , Camundongos , Camundongos SCID , MicroRNAs/biossíntese , Invasividade Neoplásica/patologia , Metástase Neoplásica/patologia , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transplante Heterólogo
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