RESUMEN
BACKGROUND: The cellular response to DNA damage is immediate and highly coordinated in order to maintain genome integrity and proper cell division. During the DNA damage response (DDR), the sensor kinases Tel1 and Mec1 in Saccharomyces cerevisiae and ATM and ATR in human, phosphorylate multiple mediators which activate effector proteins to initiate cell cycle checkpoints and DNA repair. A subset of kinase substrates are recognized by the S/T-Q cluster domain (SCD), which contains motifs of serine (S) or threonine (T) followed by a glutamine (Q). However, the full repertoire of proteins and pathways controlled by Tel1 and Mec1 is unknown. RESULTS: To identify all putative SCD-containing proteins, we analyzed the distribution of S/T-Q motifs within verified Tel1/Mec1 targets and arrived at a unifying SCD definition of at least 3 S/T-Q within a stretch of 50 residues. This new SCD definition was used in a custom bioinformatics pipeline to generate a census of SCD-containing proteins in both yeast and human. In yeast, 436 proteins were identified, a significantly larger number of hits than were expected by chance. These SCD-containing proteins did not distribute equally across GO-ontology terms, but were significantly enriched for those involved in processes related to the DDR. We also found a significant enrichment of proteins involved in telophase and cytokinesis, protein transport and endocytosis suggesting possible novel Tel1/Mec1 targets in these pathways. In the human proteome, a wide range of similar proteins were identified, including homologs of some SCD-containing proteins found in yeast. This list also included high concentrations of proteins in the Mediator, spindle pole body/centrosome and actin cytoskeleton complexes. CONCLUSIONS: Using a bioinformatic approach, we have generated a census of SCD-containing proteins that are involved not only in known DDR pathways but several other pathways under Tel1/Mec1 control suggesting new putative targets for these kinases.
Asunto(s)
Glutamina , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina , Treonina , Secuencias de Aminoácidos , Biología Computacional , Humanos , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismoRESUMEN
Point mutations of EHMT1 or deletions and duplications of chromosome 9q34.3 are found in patients with variable neurologic and developmental disorders. Here, we present a child with congenital cataract, developmental and speech delay who developed a metastatic ganglioglioma with progression to anaplastic astrocytoma. Molecular analysis identified a novel constitutional tandem duplication in 9q34.3 with breakpoints in intron 1 of TRAF2 and intron 16 of EHMT1 generating a fusion transcript predicted to encode a truncated form of EHMT1. The ganglioglioma showed complex chromosomal aberrations with further duplication of the dup9q34. Thus, this unique tandem 9q34.3 duplication may impact brain tumor formation.
Asunto(s)
Duplicación Cromosómica , Cromosomas Humanos Par 9 , Ganglioglioma/genética , N-Metiltransferasa de Histona-Lisina/genética , Preescolar , Femenino , Humanos , Factor 2 Asociado a Receptor de TNF/genéticaRESUMEN
Aberrant RNA splicing is thought to play a key role in tumorigenesis. The assessment of its specific contributions is limited by the complexity of information derived from genome-wide array-based approaches. We describe how performing splicing factor-specific comparisons using both tumor and cell line data sets may more readily identify physiologically relevant tumor-specific splicing events. Affymetrix exon array data derived from glioblastoma (GBM) tumor samples with defined polypyrimidine tract-binding protein 1 (PTBP1) levels were compared with data from U251 GBM cells with and without PTBP1 knockdown. This comparison yielded overlapping gene sets that comprised only a minor fraction of each data set. The identification of a novel GBM-specific splicing event involving the USP5 gene led us to further examine its role in tumorigenesis. In GBM, USP5 generates a shorter isoform 2 through recognition of a 5' splice site within exon 15. Production of the USP5 isoform 2 was strongly correlated with PTBP1 expression in GBM tumor samples and cell lines. Splicing regulation was consistent with the presence of an intronic PTBP1 binding site and could be modulated through antisense targeting of the isoform 2 splice site to force expression of isoform 1 in GBM cells. The forced expression of USP5 isoform 1 in two GBM cell lines inhibited cell growth and migration, implying an important role for USP5 splicing in gliomagenesis. These results support a role for aberrant RNA splicing in tumorigenesis and suggest that changes in relatively few genes may be sufficient to drive the process.
Asunto(s)
Empalme Alternativo , Endopeptidasas/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteína de Unión al Tracto de Polipirimidina/metabolismo , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Exones , Técnicas de Silenciamiento del Gen , Glioblastoma/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Datos de Secuencia Molecular , Proteína de Unión al Tracto de Polipirimidina/genética , Isoformas de Proteínas/genéticaRESUMEN
Clinical cancer genetic susceptibility analysis typically proceeds sequentially, beginning with the most likely causative gene. The process is time consuming and the yield is low, particularly for families with unusual patterns of cancer. We determined the results of in parallel mutation analysis of a large cancer-associated gene panel. We performed deletion analysis and sequenced the coding regions of 45 genes (8 oncogenes and 37 tumor suppressor or DNA repair genes) in 48 childhood cancer patients who also (i) were diagnosed with a second malignancy under age 30, (ii) have a sibling diagnosed with cancer under age 30, and/or (iii) have a major congenital anomaly or developmental delay. Deleterious mutations were identified in 6 of 48 (13%) families, 4 of which met the sibling criteria. Mutations were identified in genes previously implicated in both dominant and recessive childhood syndromes, including SMARCB1, PMS2, and TP53. No pathogenic deletions were identified. This approach has provided efficient identification of childhood cancer susceptibility mutations and will have greater utility as additional cancer susceptibility genes are identified. Integrating parallel analysis of large gene panels into clinical testing will speed results and increase diagnostic yield. The failure to detect mutations in 87% of families highlights that a number of childhood cancer susceptibility genes remain to be discovered.
Asunto(s)
Predisposición Genética a la Enfermedad , Neoplasias/epidemiología , Neoplasias/genética , Adolescente , Adulto , Algoritmos , Niño , Análisis Mutacional de ADN , Reparación del ADN , Dosificación de Gen , Genes Dominantes , Genes Recesivos , Genes Supresores de Tumor , Humanos , Inmunoprecipitación , Modelos Genéticos , Mutación , Oncogenes , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tumor Rabdoide/genéticaRESUMEN
Polypyrimidine tract-binding protein 1 (PTBP1) is a multi-functional RNA-binding protein that is aberrantly overexpressed in glioma. PTBP1 and its brain-specific homologue polypyrimidine tract-binding protein 2 (PTBP2) regulate neural precursor cell differentiation. However, the overlapping and non-overlapping target transcripts involved in this process are still unclear. To determine why PTBP1 and not PTBP2 would promote glial cell-derived tumours, both PTBP1 and PTBP2 were knocked down in the human glioma cell lines U251 and LN229 to determine the role of these proteins in cell proliferation, migration, and adhesion. Surprisingly, removal of both PTBP1 and PTBP2 slowed cell proliferation, with the double knockdown having no additive effects. Decreased expression of both proteins individually and in combination inhibited cell migration and increased adhesion of cells to fibronectin and vitronectin. A global survey of differential exon expression was performed following PTBP1 knockdown in U251 cells using the Affymetrix Exon Array to identify PTBP1-specific splicing targets that enhance gliomagenesis. In the PTBP1 knockdown, previously determined targets were unaltered in their splicing patterns. A single gene, RTN4 (Nogo) had significantly enhanced inclusion of exon 3 when PTBP1 was removed. Overexpression of the splice isoform containing exon 3 decreased cell proliferation to a similar degree as the removal of PTBP1. These results provide the first evidence that RNA-binding proteins affect the invasive and rapid growth characteristics of glioma cell lines. Its actions on proliferation appear to be mediated, in part, through alternative splicing of RTN4.
Asunto(s)
Glioma/patología , Ribonucleoproteínas Nucleares Heterogéneas/fisiología , Proteínas de Neoplasias/fisiología , Proteínas del Tejido Nervioso/fisiología , Proteína de Unión al Tracto de Polipirimidina/fisiología , Citoesqueleto de Actina/patología , Empalme Alternativo/fisiología , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proliferación Celular , Exones/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Glioma/genética , Glioma/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Humanos , Proteínas de la Mielina/biosíntesis , Proteínas de la Mielina/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nogo , Proteína de Unión al Tracto de Polipirimidina/genética , Células Tumorales CultivadasRESUMEN
BACKGROUND: Tumor-predominant splice isoforms were identified during comparative in silico sequence analysis of EST clones, suggesting that global aberrant alternative pre-mRNA splicing may be an epigenetic phenomenon in cancer. We used an exon expression array to perform an objective, genome-wide survey of glioma-specific splicing in 24 GBM and 12 nontumor brain samples. Validation studies were performed using RT-PCR on glioma cell lines, patient tumor and nontumor brain samples. RESULTS: In total, we confirmed 14 genes with glioma-specific splicing; seven were novel events identified by the exon expression array (A2BP1, BCAS1, CACNA1G, CLTA, KCNC2, SNCB, and TPD52L2). Our data indicate that large changes (> 5-fold) in alternative splicing are infrequent in gliomagenesis (< 3% of interrogated RefSeq entries). The lack of splicing changes may derive from the small number of splicing factors observed to be aberrantly expressed. CONCLUSION: While we observed some tumor-specific alternative splicing, the number of genes showing exclusive tumor-specific isoforms was on the order of tens, rather than the hundreds suggested previously by in silico mining. Given the important role of alternative splicing in neural differentiation, there may be selective pressure to maintain a majority of splicing events in order to retain glial-like characteristics of the tumor cells.
Asunto(s)
Empalme Alternativo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN/genética , Epigénesis Genética , Exones , Perfilación de la Expresión Génica , Glioma/genética , Glioma/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Polypyrimidine tract binding protein (PTB) is expressed in developing mammalian astrocytes, absent in mature adult astrocytes, and aberrantly elevated in gliomas. It is unclear whether PTB is a coincidental marker of tumor progression or a significant mediator of tumorigenesis. In developing Drosophila, the absence of the PTB homolog, hephaestus, results in increased Notch activity. Since Notch is a well-known inducer of glial cell fate, we determined whether overexpression of PTB in glial cell tumors provides a selective growth advantage by inhibiting activated Notch (Notch1IC)-mediated differentiation. To do this, we performed an immunohistochemical analysis for expression of PTB, activated Notch1 (Notch1IC), Hes1 (a Notch target), and GFAP on an extensive human tissue microarray that included 246 gliomas, 10 gliosarcomas, and 10 normal brains. Statistically significant PTB overexpression was seen in all glioma grades, with the highest increase in grade IV tumors. Notch1IC was also abnormally expressed in gliomas except in a subset of grade IV tumors in which it was absent. This decrease in Notch1IC was not associated with increased PTB expression. We conclude that PTB, and Notch1 serve as independent and functionally unlinked markers of glioma progression.