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2.
Nat Commun ; 12(1): 4535, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315877

RESUMO

Retinoblastoma is a childhood cancer of the developing retina that initiates with biallelic inactivation of the RB1 gene. Children with germline mutations in RB1 have a high likelihood of developing retinoblastoma and other malignancies later in life. Genetically engineered mouse models of retinoblastoma share some similarities with human retinoblastoma but there are differences in their cellular differentiation. To develop a laboratory model of human retinoblastoma formation, we make induced pluripotent stem cells (iPSCs) from 15 participants with germline RB1 mutations. Each of the stem cell lines is validated, characterized and then differentiated into retina using a 3-dimensional organoid culture system. After 45 days in culture, the retinal organoids are dissociated and injected into the vitreous of eyes of immunocompromised mice to support retinoblastoma tumor growth. Retinoblastomas formed from retinal organoids made from patient-derived iPSCs have molecular, cellular and genomic features indistinguishable from human retinoblastomas. This model of human cancer based on patient-derived iPSCs with germline cancer predisposing mutations provides valuable insights into the cellular origins of this debilitating childhood disease as well as the mechanism of tumorigenesis following RB1 gene inactivation.


Assuntos
Organoides/patologia , Retina/patologia , Retinoblastoma/patologia , Células-Tronco/patologia , Adulto , Diferenciação Celular , Linhagem Celular , Epigênese Genética , Éxons/genética , Feminino , Genoma Humano , Mutação em Linhagem Germinativa/genética , Humanos , Imageamento Tridimensional , Células-Tronco Pluripotentes Induzidas/metabolismo , Retinoblastoma/genética , Proteína do Retinoblastoma/genética
4.
Cancer Immunol Immunother ; 70(3): 721-732, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32915319

RESUMO

Faithful tumor mouse models are fundamental research tools to advance the field of immuno-oncology (IO). This is particularly relevant in diseases with low incidence, as in the case of pediatric malignancies, that rely on pre-clinical therapeutic development. However, conventional syngeneic and genetically engineered mouse models fail to recapitulate the tumor heterogeneity and microenvironmental complexity of human pathology that are essential determinants of cancer-directed immunity. Here, we characterize a novel mouse model that supports human natural killer (NK) cell development and engraftment of neuroblastoma orthotopic patient-derived xenograft (O-PDX) for pre-clinical antibody and cytokine testing. Using cytotoxicity assays, single-cell RNA-sequencing, and multi-color flow cytometry, we demonstrate that NK cells that develop in the humanized mice are fully licensed to execute NK cell cytotoxicity, permit human tumor engraftment, but can be therapeutically redirected to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Although these cells share phenotypic and molecular features with healthy controls, we noted that they lacked an NK cell subset, termed activated NK cells, that is characterized by differentially expressed genes that are induced by cytokine activation. Because this subset of genes is also downregulated in patients with neuroblastoma compared to healthy controls, we hypothesize that this finding could be due to tumor-mediated suppressive effects. Thus, despite its technical complexity, this humanized patient-derived xenograft mouse model could serve as a faithful system for future testing of IO applications and studies of underlying immunologic processes.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Neuroblastoma/imunologia , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Transplante de Medula Óssea , Estudos de Casos e Controles , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Feminino , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Masculino , Camundongos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nat Commun ; 11(1): 913, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060267

RESUMO

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.


Assuntos
Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/metabolismo , Proteína Nuclear Ligada ao X/genética , Animais , Pré-Escolar , Estudos de Coortes , Feminino , Amplificação de Genes , Humanos , Lactente , Masculino , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Nuclear Ligada ao X/metabolismo
6.
Cancer Cell ; 36(5): 512-527.e9, 2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31631027

RESUMO

ATRX alterations occur at high frequency in neuroblastoma of adolescents and young adults. Particularly intriguing are the large N-terminal deletions of ATRX (Alpha Thalassemia/Mental Retardation, X-linked) that generate in-frame fusion (IFF) proteins devoid of key chromatin interaction domains, while retaining the SWI/SNF-like helicase region. We demonstrate that ATRX IFF proteins are redistributed from H3K9me3-enriched chromatin to promoters of active genes and identify REST as an ATRX IFF target whose activation promotes silencing of neuronal differentiation genes. We further show that ATRX IFF cells display sensitivity to EZH2 inhibitors, due to derepression of neurogenesis genes, including a subset of REST targets. Taken together, we demonstrate that ATRX structural alterations are not loss-of-function and put forward EZH2 inhibitors as a potential therapy for ATRX IFF neuroblastoma.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Neuroblastoma/tratamento farmacológico , Proteínas Repressoras/genética , Proteína Nuclear Ligada ao X/genética , Animais , Sequência de Bases/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Feminino , Histonas/metabolismo , Humanos , Masculino , Camundongos , Neuroblastoma/genética , Neuroblastoma/patologia , Neuroblastoma/cirurgia , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Deleção de Sequência , Proteína Nuclear Ligada ao X/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Neuron ; 104(3): 512-528.e11, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31493975

RESUMO

More than 8,000 genes are turned on or off as progenitor cells produce the 7 classes of retinal cell types during development. Thousands of enhancers are also active in the developing retinae, many having features of cell- and developmental stage-specific activity. We studied dynamic changes in the 3D chromatin landscape important for precisely orchestrated changes in gene expression during retinal development by ultra-deep in situ Hi-C analysis on murine retinae. We identified developmental-stage-specific changes in chromatin compartments and enhancer-promoter interactions. We developed a machine learning-based algorithm to map euchromatin and heterochromatin domains genome-wide and overlaid it with chromatin compartments identified by Hi-C. Single-cell ATAC-seq and RNA-seq were integrated with our Hi-C and previous ChIP-seq data to identify cell- and developmental-stage-specific super-enhancers (SEs). We identified a bipolar neuron-specific core regulatory circuit SE upstream of Vsx2, whose deletion in mice led to the loss of bipolar neurons.


Assuntos
Eucromatina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Heterocromatina/metabolismo , Retina/embriologia , Células Bipolares da Retina/metabolismo , Animais , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Elementos Facilitadores Genéticos , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Aprendizado de Máquina , Camundongos , Lâmina Nuclear/metabolismo , Regiões Promotoras Genéticas , RNA-Seq , Receptores Citoplasmáticos e Nucleares/genética , Retina/citologia , Retina/metabolismo , Retina/ultraestrutura , Células Bipolares da Retina/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Análise de Célula Única , Fatores de Transcrição/genética , Receptor de Lamina B
8.
Cancer Cell ; 34(3): 411-426.e19, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30146332

RESUMO

Personalized cancer therapy targeting somatic mutations in patient tumors is increasingly being incorporated into practice. Other therapeutic vulnerabilities resulting from changes in gene expression due to tumor specific epigenetic perturbations are progressively being recognized. These genomic and epigenomic changes are ultimately manifest in the tumor proteome and phosphoproteome. We integrated transcriptomic, epigenomic, and proteomic/phosphoproteomic data to elucidate the cellular origins and therapeutic vulnerabilities of rhabdomyosarcoma (RMS). We discovered that alveolar RMS occurs further along the developmental program than embryonal RMS. We also identified deregulation of the RAS/MEK/ERK/CDK4/6, G2/M, and unfolded protein response pathways through our integrated analysis. Comprehensive preclinical testing revealed that targeting the WEE1 kinase in the G2/M pathway is the most effective approach in vivo for high-risk RMS.


Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias Musculares/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Rabdomiossarcoma/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Criança , Epigenômica , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Genômica , Humanos , Masculino , Camundongos , Terapia de Alvo Molecular/métodos , Neoplasias Musculares/genética , Neoplasias Musculares/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Medicina de Precisão/métodos , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteômica , Rabdomiossarcoma/genética , Rabdomiossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Resposta a Proteínas não Dobradas/genética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Cell Rep ; 22(10): 2601-2614, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29514090

RESUMO

Diverse cell types can be reprogrammed into pluripotent stem cells by ectopic expression of Oct4 (Pou5f1), Klf4, Sox3, and Myc. Many of these induced pluripotent stem cells (iPSCs) retain memory, in terms of DNA methylation and histone modifications (epigenetic memory), of their cellular origins, and this may bias subsequent differentiation. Neurons are difficult to reprogram, and there has not been a systematic side-by-side characterization of reprogramming efficiency or epigenetic memory across different neuronal subtypes. Here, we compare reprogramming efficiency of five different retinal cell types at two different stages of development. Retinal differentiation from each iPSC line was measured using a quantitative standardized scoring system called STEM-RET and compared to the epigenetic memory. Neurons with the lowest reprogramming efficiency produced iPSC lines with the best retinal differentiation and were more likely to retain epigenetic memory of their cellular origins. In addition, we identified biomarkers of iPSCs that are predictive of retinal differentiation.


Assuntos
Reprogramação Celular , Metilação de DNA , Histonas/metabolismo , Organogênese , Organoides/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional , Retina/citologia , Retina/metabolismo , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Núcleo Celular/metabolismo , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Camundongos , Regiões Promotoras Genéticas/genética
10.
Neuron ; 94(3): 550-568.e10, 2017 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-28472656

RESUMO

In the developing retina, multipotent neural progenitors undergo unidirectional differentiation in a precise spatiotemporal order. Here we profile the epigenetic and transcriptional changes that occur during retinogenesis in mice and humans. Although some progenitor genes and cell cycle genes were epigenetically silenced during retinogenesis, the most dramatic change was derepression of cell-type-specific differentiation programs. We identified developmental-stage-specific super-enhancers and showed that most epigenetic changes are conserved in humans and mice. To determine how the epigenome changes during tumorigenesis and reprogramming, we performed integrated epigenetic analysis of murine and human retinoblastomas and induced pluripotent stem cells (iPSCs) derived from murine rod photoreceptors. The retinoblastoma epigenome mapped to the developmental stage when retinal progenitors switch from neurogenic to terminal patterns of cell division. The epigenome of retinoblastomas was more similar to that of the normal retina than that of retina-derived iPSCs, and we identified retina-specific epigenetic memory.


Assuntos
Carcinogênese/genética , Diferenciação Celular/genética , Reprogramação Celular/genética , Metilação de DNA/genética , Epigênese Genética , Código das Histonas/genética , Retina/metabolismo , Retinoblastoma/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Retina/embriologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Proteína do Retinoblastoma/genética
11.
Nat Protoc ; 11(10): 1955-1976, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27658012

RESUMO

Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. To overcome this, other protocols have required the inactivation of the p53 tumor suppressor to reprogram postmitotic neurons, which can result in tumorigenesis of the cells. We describe a method that does not require p53 inactivation but induces reprogramming in retinal cells from reprogrammable mice grown in aggregates with wild-type mouse retinal cells. After the first 10 d of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantified using this protocol. Reprogramming retinal neurons using this protocol will take 56 d, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 d. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantifies eye field specification, optic cup formation and retinal differentiation in 3D cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol.

12.
J Mol Biol ; 428(6): 1290-1303, 2016 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-26812210

RESUMO

While the gene for p53 is mutated in many human cancers causing loss of function, many others maintain a wild-type gene but exhibit reduced p53 tumor suppressor activity through overexpression of the negative regulators, Mdm2 and/or MdmX. For the latter mechanism of loss of function, the activity of endogenous p53 can be restored through inhibition of Mdm2 or MdmX with small molecules. We previously reported a series of compounds based upon the Nutlin-3 chemical scaffold that bind to both MdmX and Mdm2 [Vara, B. A. et al. (2014) Organocatalytic, diastereo- and enantioselective synthesis of nonsymmetric cis-stilbene diamines: A platform for the preparation of single-enantiomer cis-imidazolines for protein-protein inhibition. J. Org. Chem. 79, 6913-6938]. Here we present the first solution structures based on data from NMR spectroscopy for MdmX in complex with four of these compounds and compare them with the MdmX:p53 complex. A p53-derived peptide binds with high affinity (Kd value of 150nM) and causes the formation of an extensive network of hydrogen bonds within MdmX; this constitutes the induction of order within MdmX through ligand binding. In contrast, the compounds bind more weakly (Kd values from 600nM to 12µM) and induce an incomplete hydrogen bond network within MdmX. Despite relatively weak binding, the four compounds activated p53 and induced p21(Cip1) expression in retinoblastoma cell lines that overexpress MdmX, suggesting that they specifically target MdmX and/or Mdm2. Our results document structure-activity relationships for lead-like small molecules targeting MdmX and suggest a strategy for their further optimization in the future by using NMR spectroscopy to monitor small-molecule-induced protein order as manifested through hydrogen bond formation.


Assuntos
Descoberta de Drogas/métodos , Imidazóis/química , Imidazóis/metabolismo , Piperazinas/química , Piperazinas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Relação Estrutura-Atividade
13.
Cell Stem Cell ; 17(1): 101-15, 2015 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-26140606

RESUMO

Cell-based therapies to treat retinal degeneration are now being tested in clinical trials. However, it is not known whether the source of stem cells is important for the production of differentiated cells suitable for transplantation. To test this, we generated induced pluripotent stem cells (iPSCs) from murine rod photoreceptors (r-iPSCs) and scored their ability to make retinae by using a standardized quantitative protocol called STEM-RET. We discovered that r-iPSCs more efficiently produced differentiated retinae than did embryonic stem cells (ESCs) or fibroblast-derived iPSCs (f-iPSCs). Retinae derived from f-iPSCs had fewer amacrine cells and other inner nuclear layer cells. Integrated epigenetic analysis showed that DNA methylation contributes to the defects in f-iPSC retinogenesis and that rod-specific CTCF insulator protein-binding sites may promote r-iPSC retinogenesis. Together, our data suggest that the source of stem cells is important for producing retinal neurons in three-dimensional (3D) organ cultures.


Assuntos
Epigênese Genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Retina/crescimento & desenvolvimento , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular , Reprogramação Celular , Metilação de DNA , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Retina/citologia , Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/patologia , Degeneração Retiniana/terapia
14.
Cell Rep ; 9(3): 829-41, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25437539

RESUMO

Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.


Assuntos
Reparo do DNA , Terapia de Alvo Molecular , Sarcoma de Ewing/patologia , Animais , Benzimidazóis/farmacocinética , Benzimidazóis/farmacologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Irinotecano , Camundongos Nus , Ftalazinas/farmacocinética , Ftalazinas/farmacologia , Piperazinas/farmacocinética , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Temozolomida , Ensaios Antitumorais Modelo de Xenoenxerto
15.
EMBO J ; 33(19): 2201-15, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25100735

RESUMO

Inactivation of the retinoblastoma tumor suppressor (pRb) is a common oncogenic event that alters the expression of genes important for cell cycle progression, senescence, and apoptosis. However, in many contexts, the properties of pRb-deficient cells are similar to wild-type cells suggesting there may be processes that counterbalance the transcriptional changes associated with pRb inactivation. Therefore, we have looked for sets of evolutionary conserved, functionally related genes that are direct targets of pRb/E2F proteins. We show that the expression of NANOS, a key facilitator of the Pumilio (PUM) post-transcriptional repressor complex, is directly repressed by pRb/E2F in flies and humans. In both species, NANOS expression increases following inactivation of pRb/RBF1 and becomes important for tissue homeostasis. By analyzing datasets from normal retinal tissue and pRb-null retinoblastomas, we find a strong enrichment for putative PUM substrates among genes de-regulated in tumors. These include pro-apoptotic genes that are transcriptionally down-regulated upon pRb loss, and we characterize two such candidates, MAP2K3 and MAP3K1, as direct PUM substrates. Our data suggest that NANOS increases in importance in pRb-deficient cells and helps to maintain homeostasis by repressing the translation of transcripts containing PUM Regulatory Elements (PRE).


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Proteína do Retinoblastoma/fisiologia , Animais , Animais Geneticamente Modificados , Western Blotting , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Humanos , MAP Quinase Quinase 3/genética , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 1/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Interferência de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
Pharm Res ; 31(11): 3060-72, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24906597

RESUMO

PURPOSE: Retinoblastoma is a childhood cancer of the retina. Clinical trials have shown that local delivery of broad spectrum chemotherapeutic agents is efficacious. Recent studies characterizing the genomic and epigenomic landscape of retinoblastoma identified spleen tyrosine kinase (SYK) as a promising candidate for targeted therapy. The purpose of this study was to conduct preclinical testing of the SYK antagonist R406 to evaluate it as a candidate for retinoblastoma treatment. METHODS: The efficacy of the SYK antagonist R406 delivered locally in a human orthotopic xenograft mouse model of retinoblastoma was tested. Intraocular exposure of R406 was determined for various routes and formulations. RESULTS: There was no evidence of efficacy for subconjunctival. R406. Maximal vitreal concentration was 10-fold lower than the minimal concentration required to kill retinoblastoma cells in vitro. Dosage of R406 subconjunctivally from emulsion or suspension formulations, direct intravitreal injection of the soluble prodrug of R406 (R788), and repeated topical administration of R406 all increased vitreal exposure, but failed to reach the exposure required for retinoblastoma cell death in culture. CONCLUSION: Taken together, these data suggest that R406 is not a viable clinical candidate for the treatment of retinoblastoma. This study highlights the importance of pharmacokinetic testing of molecular targeted retinoblastoma therapeutics.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Oxazinas/farmacologia , Oxazinas/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Piridinas/farmacocinética , Neoplasias da Retina/tratamento farmacológico , Retinoblastoma/tratamento farmacológico , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Olho/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Quinase Syk
17.
Springerplus ; 3: 222, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24834375

RESUMO

The SWI/SNF-like chromatin-remodeling protein ATRX has emerged as a key factor in the regulation of α-globin gene expression, incorporation of histone variants into the chromatin template and, more recently, as a frequently mutated gene across a wide spectrum of cancers. Therefore, the availability of a functional ATRX cDNA for expression studies is a valuable tool for the scientific community. We have identified two independent transposon insertions of a bacterial IS10 element into exon 8 of ATRX isoform 2 coding sequence in two different plasmids derived from a single source. We demonstrate that these insertion events are common and there is an insertion hotspot within the ATRX cDNA. Such IS10 insertions produce a truncated form of ATRX, which significantly compromises its nuclear localization. In turn, we describe ways to prevent IS10 insertion during propagation and cloning of ATRX-containing vectors, including optimal growth conditions, bacterial strains, and suggested sequencing strategies. Finally, we have generated an insertion-free plasmid that is available to the community for expression studies of ATRX.

18.
Nucleic Acids Res ; 38(12): 3963-74, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20194111

RESUMO

Numerous human pathologies result from unrepaired oxidative DNA damage. Base excision repair (BER) is responsible for the repair of oxidative DNA damage that occurs in both nuclei and mitochondria. Despite the importance of BER in maintaining genomic stability, knowledge concerning the regulation of this evolutionarily conserved repair pathway is almost nonexistent. The Saccharomyces cerevisiae BER protein, Ntg1, relocalizes to organelles containing elevated oxidative DNA damage, indicating a novel mechanism of regulation for BER. We propose that dynamic localization of BER proteins is modulated by constituents of stress response pathways. In an effort to mechanistically define these regulatory components, the elements necessary for nuclear and mitochondrial localization of Ntg1 were identified, including a bipartite classical nuclear localization signal, a mitochondrial matrix targeting sequence and the classical nuclear protein import machinery. Our results define a major regulatory system for BER which when compromised, confers a mutator phenotype and sensitizes cells to the cytotoxic effects of DNA damage.


Assuntos
Núcleo Celular/enzimologia , Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Mitocôndrias/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Transporte Ativo do Núcleo Celular , Substituição de Aminoácidos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/análise , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Sinais de Localização Nuclear , Estresse Oxidativo , Sinais Direcionadores de Proteínas , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Methods Mol Biol ; 554: 267-86, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19513680

RESUMO

Mutation of human mitochondrial DNA (mtDNA) has been linked to maternally inherited neuromuscular disorders and is implicated in more common diseases such as cancer, diabetes, and Parkinson's disease. Mutations in mtDNA also accumulate with age and are therefore believed to contribute to aging and age-related pathology. Housed within the mitochondrial matrix, mtDNA encodes several of the proteins involved in the production of ATP via the process of oxidative phosphorylation, which involves the flow of high-energy electrons through the electron transport chain (ETC). Because of its proximity to the ETC, mtDNA is highly vulnerable to oxidative damage mediated by reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals that are constantly produced by this system. Therefore, it is important to be able to measure oxidative mtDNA damage under normal physiologic conditions and during environmental or disease-associated stress. The budding yeast, Saccharomyces cerevisiae, is a facile and informative model system in which to study such mtDNA oxidative damage because it is a unicellular eukaryotic facultative anaerobe that is conditionally dependent on mitochondrial oxidative phosphorylation for viability. Here, we describe methods for quantifying oxidative mtDNA damage and mutagenesis in S. cerevisiae, several of which could be applied to the development of similar assays in mammalian cells and tissues. These methods include measuring the number of point mutations that occur in mtDNA with the erythromycin resistance assay, quantifying the amount of oxidative DNA damage utilizing a modified Southern blot assay, and measuring mtDNA integrity with the "petite induction" assay.


Assuntos
Núcleo Celular/genética , Dano ao DNA/genética , DNA Mitocondrial/genética , Mutação Puntual/genética , Saccharomyces cerevisiae/genética , Southern Blotting , Núcleo Celular/metabolismo , DNA Mitocondrial/metabolismo , Farmacorresistência Fúngica , Eritromicina , Mitocôndrias/metabolismo , Mutagênese , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/metabolismo
20.
Mol Cell Biol ; 29(3): 794-807, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19029246

RESUMO

DNAs harbored in both nuclei and mitochondria of eukaryotic cells are subject to continuous oxidative damage resulting from normal metabolic activities or environmental insults. Oxidative DNA damage is primarily reversed by the base excision repair (BER) pathway, initiated by N-glycosylase apurinic/apyrimidinic (AP) lyase proteins. To execute an appropriate repair response, BER components must be distributed to accommodate levels of genotoxic stress that may vary considerably between nuclei and mitochondria, depending on the growth state and stress environment of the cell. Numerous examples exist where cells respond to signals, resulting in relocalization of proteins involved in key biological transactions. To address whether such dynamic localization contributes to efficient organelle-specific DNA repair, we determined the intracellular localization of the Saccharomyces cerevisiae N-glycosylase/AP lyases, Ntg1 and Ntg2, in response to nuclear and mitochondrial oxidative stress. Fluorescence microscopy revealed that Ntg1 is differentially localized to nuclei and mitochondria, likely in response to the oxidative DNA damage status of the organelle. Sumoylation is associated with targeting of Ntg1 to nuclei containing oxidative DNA damage. These studies demonstrate that trafficking of DNA repair proteins to organelles containing high levels of oxidative DNA damage may be a central point for regulating BER in response to oxidative stress.


Assuntos
Núcleo Celular/enzimologia , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Mitocôndrias/enzimologia , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Compartimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Peróxido de Hidrogênio/farmacologia , Metanossulfonato de Metila/farmacologia , Mitocôndrias/efeitos dos fármacos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Oxigênio/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia
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