RESUMO
Pediatric brain tumors are the leading cause of cancer-related death in children. Patient-derived orthotopic xenografts (PDOX) of childhood brain tumors have recently emerged as a biologically faithful vehicle for testing novel and more effective therapies. Herein, we provide the histopathological and molecular analysis of 37 novel PDOX models generated from pediatric brain tumor patients treated at St. Jude Children's Research Hospital. Using a combination of histopathology, whole-genome and whole-exome sequencing, RNA-sequencing, and DNA methylation arrays, we demonstrate the overall fidelity and inter-tumoral molecular heterogeneity of pediatric brain tumor PDOX models. These models represent frequent as well as rare childhood brain tumor entities, including medulloblastoma, ependymoma, atypical teratoid rhabdoid tumor, and embryonal tumor with multi-layer rosettes. PDOX models will be valuable platforms for evaluating novel therapies and conducting pre-clinical trials to accelerate progress in the treatment of brain tumors in children. All described PDOX models and associated datasets can be explored using an interactive web-based portal and will be made freely available to the research community upon request.
Assuntos
Neoplasias Encefálicas , Modelos Animais de Doenças , Xenoenxertos , Animais , Criança , Humanos , CamundongosRESUMO
Here, we describe three new small-molecule activators of BMP signaling found by high throughput screening of a library of â¼600â¯000 small molecules. Using a cell-based luciferase assay in the BMP4-responsive human cervical carcinoma clonal cell line, C33A-2D2, we identified three compounds with similar chemotypes that each ventralize zebrafish embryos and stimulate increased expression of the BMP target genes, bmp2b and szl. Because these compounds ventralize zebrafish embryos, we have termed them "ventromorphins." As expected for a BMP pathway activator, they induce the differentiation of C2C12 myoblasts to osteoblasts. Affymetrix RNA analysis confirmed the differentiation results and showed that ventromorphins treatment elicits a genetic response similar to BMP4 treatment. Unlike isoliquiritigenin (SJ000286237), a flavone that maximally activates the pathway after 24 h of treatment, all three ventromorphins induced SMAD1/5/8 phosphorylation within 30 min of treatment and achieved peak activity within 1 h, indicating that their responses are consistent with directly activating BMP signaling.
Assuntos
Proteínas Morfogenéticas Ósseas/agonistas , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chalconas/farmacologia , Descoberta de Drogas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Proteínas Smad/metabolismo , Bibliotecas de Moléculas Pequenas/química , Peixe-Zebra/embriologiaRESUMO
Here, we report the identification of the RNA binding motif protein RBM15B/OTT3 as a new CDK11(p110) binding partner that alters the effects of CDK11 on splicing. RBM15B was initially identified as a binding partner of the Epstein-Barr virus mRNA export factor and, more recently, as a cofactor of the nuclear export receptor NXF1. In this study, we found that RBM15B co-elutes with CDK11(p110), cyclin L2α, and serine-arginine (SR) proteins, including SF2/ASF, in a large nuclear complex of â¼1-MDa molecular mass following size exclusion chromatography. Using co-immunoprecipitation experiments and in vitro pulldown assays, we mapped two distinct domains of RBM15B that are essential for its direct interaction with the N-terminal extension of CDK11(p110), cyclin L2α, and SR proteins such as 9G8 and SF2/ASF. Finally, we established that RBM15B is a functional competitor of the SR proteins SF2/ASF and 9G8, inhibits formation of the functional spliceosomal E complex, and antagonizes the positive effect of the CDK11(p110)-cyclin L2α complex on splicing both in vitro and in vivo.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/antagonistas & inibidores , Ciclinas/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Animais , Ligação Competitiva , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Fatores de Processamento de Serina-Arginina , Spliceossomos/metabolismoRESUMO
Caspase-8 is frequently deleted or silenced in neuroblastoma and other solid tumor such as medulloblastoma and small cell lung carcinoma. Caspase-8 expression can be re-established in neuroblastoma cell lines by treatment with demethylating agents or with IFN-gamma. Here we show that four different retinoic acid (RA) derivatives also increase caspase-8 protein expression in neuroblastoma, medulloblastoma and small cell lung carcinoma cell lines. This increase in protein expression is mirrored by an increase in RNA expression in NB cells. However, the promoter region of the caspase-8 gene was not responsible for the induction of caspase-8 expression. Rather, we identified another intronic region containing a CREB binding site that was required for maximal induction of caspase-8 via RA. DNA-protein interaction assays revealed increased phospho-CREB binding to this response element in RA-treated NB cells. Furthermore, mutations of the CREB binding site completely blocked caspase-8 induction in the luciferase reporter system assay and transfection of dominant-negative form of CREB repressed the up-regulation of caspase-8 by RA. Importantly, RA-released cells maintained caspase-8 expression for at least 2-5 days and were more sensitive to doxorubicin and TNFalpha. Thus, RA treatment in conjunction with TNFalpha and/or subsets of cytotoxic agents may have therapeutic benefits.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 8/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neuroblastoma/patologia , Transcrição Gênica , Tretinoína/farmacologia , Antibióticos Antineoplásicos/farmacologia , Apoptose/fisiologia , Western Blotting , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Caspase 8/metabolismo , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Metilação de DNA , Doxorrubicina/farmacologia , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Íntrons/genética , Luciferases/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/patologia , Mutação/genética , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Transdução de Sinais , Ativação Transcricional , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Although it has been reported that cyclin L1alpha and L2alpha proteins interact with CDK11(p110), the nature of the cyclin L transcripts, the formation of complexes between the five cyclin L and the three CDK11 protein isoforms, and the influence of these complexes on splicing have not been thoroughly investigated. Here we report that cyclin L1 and L2 genes generate 14 mRNA variants encoding six cyclin L proteins, one of which has not been described previously. Using cyclin L gene-specific antibodies, we demonstrate expression of multiple endogenous cyclin L proteins in human cell lines and mouse tissues. Moreover, we characterize interactions between CDK11(p110), mitosis-specific CDK11(p58), and apoptosis-specific CDK11(p46) with both cyclin Lalpha and -beta proteins and the co-elution of these proteins following size exclusion chromatography. We further establish that CDK11(p110) and associated cyclin Lalpha/beta proteins localize to splicing factor compartments and nucleoplasm and interact with serine/arginine-rich proteins. Importantly, we also determine the effect of CDK11-cyclin L complexes on pre-mRNA splicing. Preincubation of nuclear extracts with purified cyclin Lalpha and -beta isoforms depletes the extract of in vitro splicing activity. Ectopic expression of cyclin L1alpha, L1beta, L2alpha, or L2beta or active CDK11(p110) individually enhances intracellular intron splicing activity, whereas expression of CDK11(p58/p46) or kinase-dead CDK11(p110)represses splicing activity. Finally, we demonstrate that expression of cyclins Lalpha and -beta and CDK11(p110) strongly and differentially affects alternative splicing in vivo. Together, these data establish that CDK11(p110) interacts physically and functionally with cyclin Lalpha and -beta isoforms and SR proteins to regulate splicing.
Assuntos
Processamento Alternativo/fisiologia , Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Complexos Multiproteicos/metabolismo , Fatores de Transcrição/metabolismo , Núcleo Celular/genética , Quinases Ciclina-Dependentes/genética , Ciclinas/genética , Células HeLa , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Complexos Multiproteicos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/genéticaRESUMO
The Trypanosoma cruzi karyotype shows an extensive chromosomal size polymorphism. Absence of condensed mitotic chromosomes and chromatin fragility are characteristic features of T. cruzi which would allow DNA breaks and chromosomal rearrangements during cell proliferation. We have investigated by pulsed field gel electrophoresis (PFGE) eventual changes in chromosomal size during exponential and stationary phases of T. cruzi epimastigotes in culture, in G0 trypomastigotes and throughout the cell cycle in synchronized epimastigotes. T. cruzi molecular karyotype was stable throughout the cell cycle and during differentiation. Thus, the chromosomal size polymorphism previously reported in T. cruzi contrasts with the stability of the molecular karyotype observed here and suggests that chromosomal rearrangements leading to changes in chromosomal size are scarce events during the clonal propagation of this parasite.
Assuntos
Ciclo Celular/genética , Cromossomos/genética , Cromossomos/fisiologia , Instabilidade Genômica , Trypanosoma cruzi/citologia , Trypanosoma cruzi/genética , Animais , Diferenciação Celular , Proliferação de Células , Replicação do DNA , DNA de Protozoário/biossíntese , Genoma de Protozoário/genética , CariotipagemRESUMO
Loss of caspase-8 expression and resistance to cytotoxic agents occurs frequently in late stage neuroblastoma (NB). Interferon-gamma (IFN-gamma) induces caspase-8 in NB cells, sensitizing them to death receptor mediated apoptosis. This study characterizes the kinetics of this phenomenon and examines the effects of IFN-gamma on global gene expression to determine whether IFN-gamma responses are achievable at physiologically relevant doses and to define the biological effects of this cytokine. Here we examine the IFN-gamma responses of 16 NB cell lines. A single <5-min exposure to IFN-gamma (0.5 ng/ml) induced caspase-8 expression in all non-expressing cell lines and in 3/6 cell lines which already expressed high caspase-8. This increase in caspase-8 proteins was observed within 16 h and persisted for up to 9 days. Furthermore, IFN-gamma pretreatment of NB cells increased doxorubicin-induced apoptosis nearly 3-fold. Microarray analysis was used to identify additional genes involved in proliferation, signaling and apoptosis whose expression was modulated via IFN-gamma. Altered expression of these genes should further enhance the responsiveness of NB cells to chemotherapeutics. Thus, the use of IFN-gamma to sensitize NB cells to cytotoxic agents represents an attractive therapeutic strategy and warrants further investigation.
Assuntos
Regulação Neoplásica da Expressão Gênica , Interferon gama/farmacologia , Neuroblastoma/metabolismo , Apoptose , Caspase 8/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Sinergismo Farmacológico , Humanos , Metilação , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Important roles have been suggested for caspase-8, caspase-9 and Apaf-1 in controlling tumor development and their sensitivity to chemotherapeutic agents. Methylation and deletion of Apaf-1 and CASP8 results in the loss of their expression in melanoma and neuroblastoma, respectively, while CASP9 localization to 1p36.1 suggests it is a good candidate tumor suppressor. The status of CASP9 and Apaf-1 expression in numerous neuroblastoma cell lines with/without amplified MYCN and chromosome 1p36 loss-of-heterozygosity (LOH) was therefore examined to test the hypothesis that one or both of these genes are tumor suppressors in neuroblastoma. Although CASP9 is included in the region encompassing 1p36 LOH in all neuroblastoma cell lines examined, the remaining CASP9 allele(s) express a functional caspase-9 enzyme. Apaf-1 is also expressed in all neuroblastoma tumor cell lines examined. Thus, the CASP9 or Apaf-1 genes do not appear to function as tumor suppressors in MYCN amplified neuroblastomas. However, approximately 20% of the neuroblastoma cell lines with methylated CASP8 alleles are also highly resistant to staurosporine (STS)- and radiation-induced cell death, presumably because cytochrome c is not released from mitochondria. This suggests that a second, smaller sub-group of MYCN amplified neuroblastoma tumors exists with defect(s) in apoptotic signaling components upstream of caspase-9 and Apaf-1. Since no consistent differences in Bcl-2, Bcl-x(L) or Bax expression were seen in the STS- and radiation-resistant neuroblastomas, it suggests that a unique mitochondrial signaling factor(s) is responsible for the defect in cytochrome c release in this sub-group of tumors.
Assuntos
Apoptose/genética , Caspases/metabolismo , Cromossomos Humanos Par 1/genética , Genes myc/genética , Neuroblastoma/metabolismo , Proteínas/metabolismo , Fator Apoptótico 1 Ativador de Proteases , Caspase 9 , Caspases/genética , Deleção Cromossômica , Grupo dos Citocromos c/metabolismo , Primers do DNA/química , Inibidores Enzimáticos/farmacologia , Amplificação de Genes , Humanos , Immunoblotting , Hibridização in Situ Fluorescente , Perda de Heterozigosidade , Repetições de Microssatélites , Neuroblastoma/genética , Neuroblastoma/patologia , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases , Proteínas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estaurosporina/farmacologia , Transcrição Gênica , Transdução Genética , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia , Células Tumorais Cultivadas/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Proteína bcl-XRESUMO
Chromatin structure is influenced by histone modification, and this may help direct chromatin behavior to facilitate transcription, DNA replication, and DNA repair. Chromatin condensation and DNA fragmentation are the classic nuclear features but remain poorly characterized. It is highly probable that nucleosomal structure must be altered to allow these features to become apparent, but data to support this construct are lacking. We report here that in response to apoptotic signals from a death receptor (CD95 and tumor necrosis factor-alpha) or mitochondrial (staurosporine) apoptotic stimulus, the core nucleosomal histones H2A, H2B, H3, and H4 become separated from DNA during apoptosis in Jurkat and HeLa cells and are consequently detectable in the cell lysate prepared using a non-ionic detergent. The timing of this histone release from DNA correlates well with the progression of apoptosis. We also show expression of a caspase cleavage-resistant form of ICAD (ICAD-DM) in Jurkat and HeLa cells abolished DNA fragmentation and also dramatically reduced histone release in apoptotic cells. However, we demonstrate that apoptotic histone release is not an inevitable consequence of CAD/DFF-40-mediated DNA destruction as DNA fragmentation but not histone release occurs efficiently in tumor necrosis factor-alpha- and etoposide-treated NIH3T3 cells. Furthermore, in an in vitro apoptotic assay, incubation of apoptotic Jurkat cellular extract with non-apoptotic Jurkat nuclei led to nuclear DNA fragmentation without obvious histone release. Taken together, these data demonstrate that CAD/DFF-40 functions indirectly in mediating nucleosomal destruction during apoptosis.