Asunto(s)
Biotecnología/historia , Biología Molecular/historia , Agricultura/historia , Animales , Bovinos , Clonación Molecular , ADN Complementario/genética , Hormona del Crecimiento/historia , Hormona del Crecimiento/aislamiento & purificación , Historia del Siglo XX , Humanos , Israel , Poli A/química , Poli A/aislamiento & purificación , ARN Mensajero/química , ARN Mensajero/aislamiento & purificaciónRESUMEN
Glioblastomas (GBM) grow in a rich neurochemical milieu, but the impact of neurochemicals on GBM growth is largely unexplored. We interrogated 680 neurochemical compounds in patient-derived GBM neural stem cells (GNS) to determine the effects on proliferation and survival. Compounds that modulate dopaminergic, serotonergic, and cholinergic signaling pathways selectively affected GNS growth. In particular, dopamine receptor D4 (DRD4) antagonists selectively inhibited GNS growth and promoted differentiation of normal neural stem cells. DRD4 antagonists inhibited the downstream effectors PDGFRß, ERK1/2, and mTOR and disrupted the autophagy-lysosomal pathway, leading to accumulation of autophagic vacuoles followed by G0/G1 arrest and apoptosis. These results demonstrate a role for neurochemical pathways in governing GBM stem cell proliferation and suggest therapeutic approaches for GBM.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Células-Madre Neurales/efectos de los fármacos , Receptores de Dopamina D4/metabolismo , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Animales , Autofagia , Neoplasias Encefálicas/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/metabolismo , Humanos , Ratones , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/efectos de los fármacos , Células-Madre Neurales/citología , Células-Madre Neurales/patología , Receptores de Dopamina D4/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Análisis de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Functional heterogeneity within tumors presents a significant therapeutic challenge. Here we show that quiescent, therapy-resistant Sox2(+) cells propagate sonic hedgehog subgroup medulloblastoma by a mechanism that mirrors a neurogenic program. Rare Sox2(+) cells produce rapidly cycling doublecortin(+) progenitors that, together with their postmitotic progeny expressing NeuN, comprise tumor bulk. Sox2(+) cells are enriched following anti-mitotic chemotherapy and Smoothened inhibition, creating a reservoir for tumor regrowth. Lineage traces from Sox2(+) cells increase following treatment, suggesting that this population is responsible for relapse. Targeting Sox2(+) cells with the antineoplastic mithramycin abrogated tumor growth. Addressing functional heterogeneity and eliminating Sox2(+) cells presents a promising therapeutic paradigm for treatment of sonic hedgehog subgroup medulloblastoma.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Proliferación Celular , Neoplasias Cerebelosas/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , Factores de Transcripción SOXB1/metabolismo , Animales , Antígenos Nucleares/metabolismo , Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Linaje de la Célula , Proliferación Celular/efectos de los fármacos , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Proteínas de Unión al ADN , Relación Dosis-Respuesta a Droga , Proteínas de Dominio Doblecortina , Resistencia a Antineoplásicos , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog/genética , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Recurrencia Local de Neoplasia , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis , Neuropéptidos/metabolismo , Proteínas Nucleares/metabolismo , Receptores Patched , Plicamicina/farmacología , Pronóstico , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Factores de Transcripción SOXB1/genética , Receptor Smoothened , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The SAM domain of the Saccharomyces cerevisiae post-transcriptional regulator Vts1p epitomizes a subfamily of SAM domains conserved from yeast to humans that function as sequence-specific RNA-binding domains. Here we report the 2.0-A X-ray structure of the Vts1p SAM domain bound to a high-affinity RNA ligand. Specificity of RNA binding arises from the association of a guanosine loop base with a shallow pocket on the SAM domain and from multiple SAM domain contacts to the unique backbone structure of the loop, defined in part by a nonplanar base pair within the loop. We have validated NNF1 as an endogenous target of Vts1p among 79 transcripts that copurify with Vts1p. Bioinformatic analysis of these mRNAs demonstrates that the RNA-binding specificity of Vts1p in vivo is probably more stringent than that of the isolated SAM domain in vitro.
Asunto(s)
Conformación de Ácido Nucleico , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Emparejamiento Base , Sitios de Unión , Cristalografía por Rayos X , Internet , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , ARN de Hongos/química , Proteínas de Unión al ARN/genética , Elementos de Respuesta/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
The SAM domain of the Saccharomyces cerevisiae post-transcriptional regulator Vts1 has a high affinity towards RNA hairpins containing a CUGGC pentaloop. We present the 1.6 Angstroms X-ray crystal structure of the Vts1 SAM domain in its unliganded state, and the NMR solution structure of this domain in its RNA-bound state. Both structures reveal a canonical five helix SAM domain flanked by additional secondary structural elements at the N and C termini. The two structures are essentially identical, implying that no major structural rearrangements occur upon RNA binding. Amide chemical shift changes map the RNA-binding site to a shallow, basic patch at the junction of helix alpha5 and the loop connecting helices alpha1 and alpha2.
Asunto(s)
Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , ARN/química , Proteínas de Saccharomyces cerevisiae/química , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Anteroposterior patterning in Drosophila melanogaster is dependent on the sequence-specific RNA-binding protein Smaug, which binds to and regulates the translation of nanos (nos) mRNA. Here we demonstrate that the sterile-alpha motif (SAM) domain of Smaug functions as an RNA-recognition domain. This represents a new function for the SAM domain family, which is well characterized for mediating protein-protein interactions. Using homology modeling and site-directed mutagenesis, we have localized the RNA-binding surface of the Smaug SAM domain and have elaborated the RNA consensus sequence required for binding. Residues that compose the RNA-binding surface are conserved in a subgroup of SAM domain-containing proteins, suggesting that the function of the domain is conserved from yeast to humans. We show here that the SAM domain of Saccharomyces cerevisiae Vts1 binds RNA with the same specificity as Smaug and that Vts1 induces transcript degradation through a mechanism involving the cytoplasmic deadenylase CCR4. Together, these results suggest that Smaug and Vts1 define a larger class of post-transcriptional regulators that act in part through a common transcript-recognition mechanism.