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1.
J Pharmacol Exp Ther ; 387(3): 315-327, 2023 12.
Artículo en Inglés | MEDLINE | ID: mdl-37827699

RESUMEN

Achieving adequate exposure of the free therapeutic agent at the target is a critical determinant of efficacious chemotherapy. With this in mind, a major challenge in developing therapies for central nervous system (CNS) tumors is to overcome barriers to delivery, including the blood-brain barrier (BBB). Panobinostat is a nonselective pan-histone deacetylase inhibitor that is being tested in preclinical and clinical studies, including for the treatment of pediatric medulloblastoma, which has a propensity for leptomeningeal spread and diffuse midline glioma, which can infiltrate into supratentorial brain regions. In this study, we examined the rate, extent, and spatial heterogeneity of panobinostat CNS distribution in mice. Transporter-deficient mouse studies show that panobinostat is a dual substrate of P-glycoprotein (P-gp) and breast cancer resistant protein (Bcrp), which are major efflux transporters expressed at the BBB. The CNS delivery of panobinostat was moderately limited by P-gp and Bcrp, and the unbound tissue-to-plasma partition coefficient of panobinostat was 0.32 and 0.21 in the brain and spinal cord in wild-type mice. In addition, following intravenous administration, panobinostat demonstrated heterogeneous distribution among brain regions, indicating that its efficacy would be influenced by tumor location or the presence and extent of leptomeningeal spread. Simulation using a compartmental BBB model suggests inadequate exposure of free panobinostat in the brain following a recommended oral dosing regimen in patients. Therefore, alternative approaches to CNS delivery may be necessary to have adequate exposure of free panobinostat for the treatment of a broad range of pediatric brain tumors. SIGNIFICANCE STATEMENT: This study shows that the central nervous system (CNS) penetration of panobinostat is limited by P-gp and Bcrp, and its efficacy may be limited by inadequate distribution to the tumor. Panobinostat has heterogeneous distribution into various brain regions, indicating that its efficacy might depend on the anatomical location of the tumors. These distributional parameters in the mouse CNS can inform both preclinical and clinical trial study design and may guide treatment for these devastating brain tumors in children.


Asunto(s)
Transportadoras de Casetes de Unión a ATP , Neoplasias Encefálicas , Niño , Humanos , Animales , Ratones , Panobinostat/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Proteínas de Neoplasias/metabolismo , Sistema Nervioso Central/metabolismo , Encéfalo/metabolismo , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Proteínas de Transporte de Membrana/metabolismo
2.
Artículo en Inglés | MEDLINE | ID: mdl-37582863

RESUMEN

BACKGROUND: Self-stigma among people with mental illness is negatively associated with personal and clinical recovery. Due to the concealable nature of mental illness, people with mental illness experience constant struggles between concealment and disclosure. Disclosure of mental health challenges can potentially minimize negative impacts of self-stigma and enhance self-esteem and sense of empowerment. Honest, Open, Proud (HOP) is a peer-led intervention that promotes autonomous and dignified decisions about disclosure. PURPOSE: This study examined the effectiveness of HOP on concealment motivation, empowerment, self-stigma, stigma stress, and recovery among people with lived experience of mental illness in Hong Kong. METHODOLOGY: A total of 162 participants with a mean age of 45.38 were recruited and randomized into intervention group and waitlist control group. Participants in the intervention group were invited to attend a 6-session HOP group intervention. RESULTS: Significant improvement in optimism score from the empowerment scale was found in the intervention group compared to the waitlist control group and the effect was sustained at 1-month follow-up. However, significant changes were not found in other outcome variables. CONCLUSION: Only improvement in optimism was observed in the current study. Future study needs to examine the effects of HOP with further modification to maximize the benefit for people with lived experience of mental illness in the local context.

3.
Bioinformatics ; 36(4): 1270-1272, 2020 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-31566663

RESUMEN

MOTIVATION: The traditional reads per million normalization method is inappropriate for the evaluation of ChIP-seq data when treatments or mutations have global effects. Changes in global levels of histone modifications can be detected with exogenous reference spike-in controls. However, most ChIP-seq studies overlook the normalization that must be corrected with spike-in. A method that retrospectively renormalizes datasets without spike-in is lacking. RESULTS: ChIPseqSpikeInFree is a novel ChIP-seq normalization method to effectively determine scaling factors for samples across various conditions and treatments, which does not rely on exogenous spike-in chromatin or peak detection to reveal global changes in histone modification occupancy. Application of ChIPseqSpikeInFree on five datasets demonstrates that this in silico approach reveals a similar magnitude of global changes as the spike-in method does. AVAILABILITY AND IMPLEMENTATION: St. Jude Cloud (https://pecan.stjude.cloud/permalink/spikefree) and St. Jude Github ( https://github.com/stjude/ChIPseqSpikeInFree). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Código de Histonas , Cromatina , Inmunoprecipitación de Cromatina , Estudios Retrospectivos , Análisis de Secuencia de ADN
4.
Acta Neuropathol ; 137(4): 637-655, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30770999

RESUMEN

Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27M-dependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression signatures from K27M primary DIPG tumors and are strongly enriched for genes associated with nervous system development. Integrated analysis of ChIP-seq and expression data showed that genes upregulated by the mutation are overrepresented in apparently bivalent promoters. Many of these targets are associated with more immature differentiation states. Expression profiles indicate K27M knockdown decreases proliferation and increases differentiation within lineages represented in DIPG. These data suggest that K27M-mediated loss of H3K27me3 directly regulates a subset of genes by releasing poised promoters, and contributes to tumor phenotype and growth by limiting differentiation. The delayed tumor growth associated with knockdown of H3 K27M provides evidence that this highly recurrent mutation is a relevant therapeutic target.


Asunto(s)
Neoplasias del Tronco Encefálico/genética , Diferenciación Celular/genética , Glioma Pontino Intrínseco Difuso/genética , Histonas/genética , Mutación , Animales , Neoplasias del Tronco Encefálico/patología , Línea Celular Tumoral , Glioma Pontino Intrínseco Difuso/patología , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Ratones
6.
Development ; 139(4): 793-804, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22274699

RESUMEN

The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.


Asunto(s)
Región Branquial/embriología , Pronefro/embriología , Factores de Transcripción/genética , Proteínas de Pez Cebra/genética , Pez Cebra/anatomía & histología , Pez Cebra/embriología , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Evolución Biológica , Biomarcadores/metabolismo , Región Branquial/metabolismo , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/fisiología , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Riñón/anatomía & histología , Morfogénesis/fisiología , Factor de Transcripción PAX2/genética , Factor de Transcripción PAX2/metabolismo , Fenotipo , Pronefro/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo
7.
Development ; 137(18): 3119-28, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20736288

RESUMEN

Endothelial tubulogenesis is a crucial step in the formation of functional blood vessels during angiogenesis and vasculogenesis. Here, we use in vivo imaging of living zebrafish embryos expressing fluorescent fusion proteins of beta-Actin, alpha-Catenin, and the ERM family member Moesin1 (Moesin a), to define a novel cord hollowing process that occurs during the initial stages of tubulogenesis in intersegmental vessels (ISVs) in the embryo. We show that the primary lumen elongates along cell junctions between at least two endothelial cells during embryonic angiogenesis. Moesin1-EGFP is enriched around structures that resemble intracellular vacuoles, which fuse with the luminal membrane during expansion of the primary lumen. Analysis of silent heart mutant embryos shows that initial lumen formation in the ISVs is not dependent on blood flow; however, stabilization of a newly formed lumen is dependent upon blood flow. Zebrafish moesin1 knockdown and cell transplantation experiments demonstrate that Moesin1 is required in the endothelial cells of the ISVs for in vivo lumen formation. Our analyses suggest that Moesin1 contributes to the maintenance of apical/basal cell polarity of the ISVs as defined by adherens junctions. Knockdown of the adherens junction protein Ve-cadherin disrupts formation of the apical membrane and lumen in a cell-autonomous manner. We suggest that Ve-cadherin and Moesin1 function to establish and maintain apical/basal polarity during multicellular lumen formation in the ISVs.


Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Embrión no Mamífero/irrigación sanguínea , Embrión no Mamífero/metabolismo , Células Endoteliales/metabolismo , Proteínas de Microfilamentos/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Uniones Adherentes/metabolismo , Animales , Antígenos CD/genética , Cadherinas/genética , Polaridad Celular , Células Endoteliales/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Neovascularización Fisiológica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Pez Cebra/genética , Proteína de la Zonula Occludens-1
8.
Cancer Res ; 83(1): 130-140, 2023 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-36264168

RESUMEN

Deregulation of neuroblastoma-derived myc (N-myc) is a leading cause of malignant brain tumors in children. To target N-myc-driven medulloblastoma, most research has focused on identifying genomic alterations or on the analysis of the medulloblastoma transcriptome. Here, we have broadly characterized the translatome of medulloblastoma and shown that N-myc unexpectedly drives selective translation of transcripts that promote protein homeostasis. Cancer cells are constantly exposed to proteotoxic stress associated with alterations in protein production or folding. It remains poorly understood how cancers cope with proteotoxic stress to promote their growth. Here, our data revealed that N-myc regulates the expression of specific components (∼5%) of the protein folding machinery at the translational level through the major cap binding protein, eukaryotic initiation factor eIF4E. Reducing eIF4E levels in mouse models of medulloblastoma blocked tumorigenesis. Importantly, targeting Hsp70, a protein folding chaperone translationally regulated by N-myc, suppressed tumor growth in mouse and human medulloblastoma xenograft models. These findings reveal a previously hidden molecular program that promotes medulloblastoma formation and identify new therapies that may have impact in the clinic. SIGNIFICANCE: Translatome analysis in medulloblastoma shows that N-myc drives selective translation of transcripts that promote protein homeostasis and that represent new therapeutic vulnerabilities.


Asunto(s)
Neoplasias Cerebelosas , Meduloblastoma , Niño , Humanos , Ratones , Animales , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Meduloblastoma/patología , Factor 4E Eucariótico de Iniciación/genética , Modelos Animales de Enfermedad , Neoplasias Cerebelosas/patología
9.
Nat Genet ; 55(12): 2189-2199, 2023 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-37945900

RESUMEN

Circular extrachromosomal DNA (ecDNA) in patient tumors is an important driver of oncogenic gene expression, evolution of drug resistance and poor patient outcomes. Applying computational methods for the detection and reconstruction of ecDNA across a retrospective cohort of 481 medulloblastoma tumors from 465 patients, we identify circular ecDNA in 82 patients (18%). Patients with ecDNA-positive medulloblastoma were more than twice as likely to relapse and three times as likely to die within 5 years of diagnosis. A subset of tumors harbored multiple ecDNA lineages, each containing distinct amplified oncogenes. Multimodal sequencing, imaging and CRISPR inhibition experiments in medulloblastoma models reveal intratumoral heterogeneity of ecDNA copy number per cell and frequent putative 'enhancer rewiring' events on ecDNA. This study reveals the frequency and diversity of ecDNA in medulloblastoma, stratified into molecular subgroups, and suggests copy number heterogeneity and enhancer rewiring as oncogenic features of ecDNA.


Asunto(s)
Neoplasias Cerebelosas , Meduloblastoma , Neoplasias , Humanos , ADN Circular , Meduloblastoma/genética , Estudios Retrospectivos , Neoplasias/genética , Oncogenes , Neoplasias Cerebelosas/genética
10.
Int J Psychiatry Clin Pract ; 16(4): 284-92, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22217497

RESUMEN

OBJECTIVE: A large-scale quantitative study was conducted by stratified representative samples from Chicago (prototype of the United States; N = 293), Beijing (prototype of urbanized China; N = 302), and Hong Kong (prototype of East-meets-West culture; N = 284) to explore factors that might lead to their stigmatizing attitudes towards hiring individuals with (mental illness, alcohol abuse, drug abuse, and HIV/AIDS) and without (bone cancer) behavioral-driven health conditions. METHODS: Consented employers completed the Employer Survey pertaining to their attitudes towards specific health conditions, previous hiring experiences, resources, assets of applicants, and hiring concerns. RESULTS: The findings suggested that employers in Hong Kong and Beijing were more willing to hire individuals with alcohol abuse, whereas employers in Chicago were more willing to hire those with HIV/AIDS or bone cancer. Logistic regression suggested that the type of health conditions, assets of applicants, and perceived level of dangerousness of applicants were significant predictors that contributed to employers' hiring preference. CONCLUSION: Employers express different hiring preference towards individuals with or without behavioral-driven health conditions. Their hiring preference towards specific type of health conditions is discussed.


Asunto(s)
Actitud Frente a la Salud , Empleo/psicología , Trastornos Mentales/psicología , Selección de Personal/estadística & datos numéricos , Estereotipo , Neoplasias Óseas/psicología , Chicago , China , Cultura , Empleo/estadística & datos numéricos , Femenino , Infecciones por VIH/psicología , Humanos , Masculino , Persona de Mediana Edad , Satisfacción Personal , Análisis de Regresión
11.
Biomaterials ; 280: 121276, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34890975

RESUMEN

Genetic screens are powerful tools for both resolving biological function and identifying potential therapeutic targets, but require physiologically accurate systems to glean biologically useful information. Here, we enable genetic screens in physiologically relevant ex vivo cancer tissue models by integrating CRISPR-Cas-based genome engineering and biofabrication technologies. We first present a novel method for generating perfusable tissue constructs, and validate its functionality by using it to generate three-dimensional perfusable dense cultures of cancer cell lines and sustain otherwise ex vivo unculturable patient-derived xenografts. Using this system we enable large-scale CRISPR screens in perfused tissue cultures, as well as emulate a novel point-of-care diagnostics scenario of a clinically actionable CRISPR knockout (CRISPRko) screen of genes with FDA-approved drug treatments in ex vivo PDX cell cultures. Our results reveal differences across in vitro and in vivo cancer model systems, and highlight the utility of programmable tissue engineered models for screening therapeutically relevant cancer vulnerabilities.


Asunto(s)
Neoplasias , Ingeniería de Tejidos , Sistemas CRISPR-Cas/genética , Detección Precoz del Cáncer , Genoma , Humanos , Neoplasias/genética
12.
Appl Neuropsychol Adult ; : 1-8, 2021 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-34860619

RESUMEN

It is well established that long-term postconcussive symptoms following a mild traumatic brain injury (mTBI) are associated with underlying physical, emotional, and behavioral conditions. The Neurobehavioral Symptom Inventory (NSI) is a measure used to assess neurobehavioral symptoms that can occur following a mTBI and has demonstrated a 3- or 4-factor structure in veterans. The present study aimed to investigate the factor structure of veterans with PTSD without a history of mTBI. A confirmatory factor analysis (CFA) was conducted on a sample of 221 treatment-seeking veterans and service members with PTSD and without a history of mTBI. Results supported a 4-factor structure comprised of vestibular, somatic, cognitive, and affective domains in veterans with PTSD. Subsequent, correlational analyses between the four NSI factors and the four subscales of the Posttraumatic Stress Disorder Checklist - Fifth Edition (PCL-5) revealed high correspondence between the cognitive and affective factors of the NSI and the negative alterations in mood and cognitions and hyperarousal symptom subscales of PTSD. Collectively, findings demonstrated that the NSI functions similarly in veterans with PTSD with or without a history of mTBI. Findings suggest that neurobehavioral symptoms assessed by the NSI appear to be nonspecific and not explicitly associated with mTBI.

13.
J Mol Cell Cardiol ; 49(2): 287-93, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20206635

RESUMEN

Heparan sulfate proteoglycans are abundant molecules in the extracellular matrix and at the cell surface. Heparan sulfate chains are composed of groups of disaccharides whose side chains are modified through a series of enzymatic reactions. Deletion of these enzymes alters heparan sulfate fine structure and leads to changes in cell proliferation and tissue development. The role of heparan sulfate modification has not been explored in the vessel wall. The goal of this study was to test the hypothesis that altering heparan sulfate fine structure would impact vascular smooth muscle cell (VSMC) proliferation, vessel structure, and remodeling in response to injury. A heparan sulfate modifying enzyme, N-deacetylase N-sulfotransferase1 (Ndst1) was deleted in smooth muscle resulting in decreased N- and 2-O sulfation of the heparan sulfate chains. Smooth muscle specific deletion of Ndst1 led to a decrease in proliferating VSMCs and the circumference of the femoral artery in neonatal and adult mice. In response to vascular injury, mice lacking Ndst1 exhibited a significant reduction in lesion formation. Taken together, these data provide new evidence that modification of heparan sulfate fine structure through deletion of Ndst1 is sufficient to decrease VSMC proliferation and alter vascular remodeling.


Asunto(s)
Vasos Sanguíneos/patología , Vasos Sanguíneos/fisiopatología , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/enzimología , Miocitos del Músculo Liso/patología , Sulfotransferasas/deficiencia , Envejecimiento/patología , Animales , Animales Recién Nacidos , Vasos Sanguíneos/enzimología , Proliferación Celular , Arteria Femoral/enzimología , Arteria Femoral/patología , Eliminación de Gen , Pruebas de Función Cardíaca , Heparitina Sulfato/metabolismo , Ratones , Tamaño de los Órganos , Sulfotransferasas/metabolismo , Túnica Íntima/enzimología , Túnica Íntima/patología , Túnica Íntima/fisiopatología , Túnica Media/enzimología , Túnica Media/patología , Túnica Media/fisiopatología
14.
PLoS Genet ; 3(5): e78, 2007 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-17530925

RESUMEN

Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected through the transferase-mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off-targeting. Importantly, reversal of p53-dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse-transcriptase PCR, microarrays and whole-mount in situ hybridization assays show that MO off-targeting effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies.


Asunto(s)
Técnicas Genéticas , Activación Transcripcional , Proteína p53 Supresora de Tumor/genética , Pez Cebra/genética , Animales , Apoptosis/efectos de los fármacos , Artefactos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Morfolinas/farmacología , Morfolinos , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fenotipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sensibilidad y Especificidad , Especificidad por Sustrato , Factores de Tiempo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/embriología
15.
Methods Mol Biol ; 1869: 207-230, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30324526

RESUMEN

To maximize the physiological relevance of in vivo brain tumor mouse models designed to study the downstream effects of oncogenic mutations, it is important to express the mutated genes at appropriate levels, in relevant cell types, and in the proper developmental context. For recurrent mutations found in the heterozygous state in tumors, expression of the mutation from the endogenous locus is a more physiologically relevant recapitulation of the brain tumor genome. Here, we describe an approach to generate knock-in mice with an inducible mutation recombined into the endogenous locus. In these engineered mice, the mutated allele is designed for expression controlled by the endogenous promoter and regulatory elements after Cre recombinase-mediated deletion of a loxP-STOP-loxP cassette inserted upstream of the translational start site. To preserve the structure of the endogenous locus, mutations or additional elements may need to be inserted at a considerable distance from the loxP-STOP-loxP cassette. We used recombineering to build a construct with two selectable markers and multiple genetic alterations that can be introduced into the endogenous allele in cis with a single ES cell targeting.


Asunto(s)
Neoplasias Encefálicas/genética , Técnicas de Sustitución del Gen/métodos , Ingeniería Genética/métodos , Sitios Genéticos , Mutación/genética , Oncogenes , Animales , Cromosomas Artificiales Bacterianos/genética , Modelos Animales de Enfermedad , Electroporación , Vectores Genéticos/metabolismo , Genómica , Recombinación Homóloga/genética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Mutación Puntual/genética
16.
Cancer Cell ; 35(1): 140-155.e7, 2019 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-30595505

RESUMEN

Diffuse intrinsic pontine gliomas (DIPGs) are incurable childhood brainstem tumors with frequent histone H3 K27M mutations and recurrent alterations in PDGFRA and TP53. We generated genetically engineered inducible mice and showed that H3.3 K27M enhanced neural stem cell self-renewal while preserving regional identity. Neonatal induction of H3.3 K27M cooperated with activating platelet-derived growth factor receptor α (PDGFRα) mutant and Trp53 loss to accelerate development of diffuse brainstem gliomas that recapitulated human DIPG gene expression signatures and showed global changes in H3K27 posttranslational modifications, but relatively restricted gene expression changes. Genes upregulated in H3.3 K27M tumors were enriched for those associated with neural development where H3K27me3 loss released the poised state of apparently bivalent promoters, whereas downregulated genes were enriched for those encoding homeodomain transcription factors.


Asunto(s)
Neoplasias del Tronco Encefálico/genética , Perfilación de la Expresión Génica/métodos , Glioma/genética , Histonas/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Proteína p53 Supresora de Tumor/genética , Animales , Autorrenovación de las Células , Células Cultivadas , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , Ratones , Mutación , Células-Madre Neurales/citología , Rombencéfalo/patología , Análisis de Secuencia de ARN/métodos
17.
Cancer Res ; 79(5): 905-917, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30674530

RESUMEN

Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. SIGNIFICANCE: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.


Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Cerebelosas/genética , Meduloblastoma/genética , Tumores Neuroectodérmicos Primitivos/genética , Animales , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Transformación Celular Neoplásica/genética , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Elementos Transponibles de ADN/genética , Femenino , Factores de Transcripción Forkhead/genética , Proteínas Activadoras de GTPasa/biosíntesis , Proteínas Activadoras de GTPasa/genética , Humanos , Masculino , Meduloblastoma/metabolismo , Meduloblastoma/patología , Proteínas de la Membrana/genética , Ratones , Ratones Desnudos , Mutagénesis Insercional/métodos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Tumores Neuroectodérmicos Primitivos/metabolismo , Tumores Neuroectodérmicos Primitivos/patología , Pronóstico
18.
Psychiatr Serv ; 69(4): 456-461, 2018 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-29241431

RESUMEN

OBJECTIVE: The impact of a peer navigator program (PNP) on efforts to address the health needs of Latinos with serious mental illness was examined in a randomized controlled trial. METHODS: Latinos with a serious mental illness (N=110) were randomly assigned to the PNP (integrated care with a peer navigator [PN]) or to a treatment-as-usual control group (integrated care without a PN) for one year. Data on service engagement (scheduled and received appointments) were assessed weekly, and self-reports of recovery, empowerment, and quality of life were collected at baseline and at four, eight, and 12 months. RESULTS: Findings from group × trial analyses of covariance (ANCOVAs) found main and interaction effects for scheduled and achieved appointments, showing better engagement for the PNP group compared with the control group over the course of the study. Significant interactions were found for recovery, empowerment, and quality of life, showing greater improvement for the PNP group compared with the control group over year 1 of the study (multivariate ANCOVA; F=3.27, df=9 and 98, p<.01). CONCLUSIONS: In-the-field navigation by peers seems to enhance service engagement, recovery, and quality of life. Whether these results occurred because navigators helped overcome barriers to treatment-regardless of whether they were peers per se-needs to be examined in future research.


Asunto(s)
Hispánicos o Latinos/estadística & datos numéricos , Trastornos Mentales/terapia , Servicios de Salud Mental/estadística & datos numéricos , Evaluación de Procesos y Resultados en Atención de Salud/estadística & datos numéricos , Navegación de Pacientes/estadística & datos numéricos , Participación del Paciente/estadística & datos numéricos , Grupo Paritario , Adulto , Chicago , Prestación Integrada de Atención de Salud , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Calidad de Vida
19.
Cancer Res ; 77(1): 123-133, 2017 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-27815386

RESUMEN

Loss of the tumor suppressor gene PTEN exerts diverse outcomes on cancer in different developmental contexts. To gain insight into the effect of its loss on outcomes in the brain, we conditionally inactivated the murine Pten gene in neonatal neural stem/progenitor cells. Pten inactivation created an abnormal perivascular proliferative niche in the cerebellum that persisted in adult animals but did not progress to malignancy. Proliferating cells showed undifferentiated morphology and expressed the progenitor marker Nestin but not Math1, a marker of committed granule neuron progenitors. Codeletion of Pten and Trp53 resulted in fully penetrant medulloblastoma originating from the perivascular niche, which exhibited abnormal blood vessel networks and advanced neuronal differentiation of tumor cells. EdU pulse-chase experiments demonstrated a perivascular cancer stem cell population in Pten/Trp53 double mutant medulloblastomas. Genetic analyses revealed recurrent somatic inactivations of the tumor suppressor gene Ptch1 and a recapitulation of the sonic hedgehog subgroup of human medulloblastomas. Overall, our results showed that PTEN acts to prevent the proliferation of a progenitor niche in postnatal cerebellum predisposed to oncogenic induction of medulloblastoma. Cancer Res; 77(1); 123-33. ©2016 AACR.


Asunto(s)
Neoplasias Cerebelosas/genética , Meduloblastoma/genética , Células Madre Neoplásicas/patología , Células-Madre Neurales/patología , Fosfohidrolasa PTEN/genética , Animales , Neoplasias Cerebelosas/patología , Hibridación Genómica Comparativa , Modelos Animales de Enfermedad , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Meduloblastoma/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Transducción de Señal/fisiología , Nicho de Células Madre/fisiología , Proteína p53 Supresora de Tumor/genética
20.
BMC Mol Biol ; 7: 36, 2006 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-17042952

RESUMEN

BACKGROUND: Loss of heterozygosity (LOH) contributes to many cancers, but the rate at which these events occur in normal cells of the body is not clear. LOH would be detectable in diverse cell types in the body if this event were to confer an obvious cellular phenotype. Mice that carry two different fluorescent protein genes as alleles of a locus would seem to be a useful tool for addressing this issue because LOH would change a cell's phenotype from dichromatic to monochromatic. In addition, LOH caused by mitotic crossing over might be discernable in tissues because this event produces a pair of neighboring monochromatic cells that are different colors. RESULTS: As a step in assessing the utility of this approach, we derived primary embryonic fibroblast populations and embryonic stem cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. Fluorescence activated cell sorting (FACS) showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, and that populations exhibited expression noise similar to that seen in bacteria and yeast. Cells with a monochromatic phenotype were present at frequencies on the order of 10(-4) and appeared to be produced at a rate of approximately 10(-5) variant cells per mitosis. 45 of 45 stably monochromatic ES cell clones exhibited loss of the expected allele at the ROSA26 locus. More than half of these clones retained heterozygosity at a locus between ROSA26 and the centromere. Other clones exhibited LOH near the centromere, but were disomic for chromosome 6. CONCLUSION: Allelic fluorescent markers allowed LOH at the ROSA26 locus to be detected by FACS. LOH at this locus was usually not accompanied by LOH near the centromere, suggesting that mitotic recombination was the major cause of ROSA26 LOH. Dichromatic mouse embryonic cells provide a novel system for studying genetic/karyotypic stability and factors influencing expression from allelic genes. Similar approaches will allow these phenomena to be studied in tissues.


Asunto(s)
Proteínas Bacterianas/biosíntesis , Fibroblastos/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Pérdida de Heterocigocidad/genética , Proteínas Luminiscentes/biosíntesis , Proteínas/genética , Alelos , Animales , Proteínas Bacterianas/genética , Células Cultivadas/metabolismo , Centrómero/ultraestructura , Pintura Cromosómica , Metanosulfonato de Etilo/farmacología , Citometría de Flujo , Eliminación de Gen , Dosificación de Gen , Expresión Génica , Marcadores Genéticos , Inestabilidad Genómica , Proteínas Fluorescentes Verdes/genética , Heterocigoto , Proteínas Luminiscentes/genética , Masculino , Ratones , Ratones Transgénicos , Repeticiones de Microsatélite , Mitosis , Monosomía , Mutación , Fenotipo , ARN no Traducido , Recombinación Genética , Trisomía
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