RESUMEN
BACKGROUND: The phosphatase chronophin (CIN/PDXP) has been shown to be an important regulator of glioma cell migration and invasion. It has two known substrates: p-Ser3-cofilin, the phosphorylated form of the actin binding protein cofilin, and pyridoxal 5'-phosphate, the active form of vitamin B6. Phosphoregulation of cofilin, among other functions, plays an important role in cell migration, whereas active vitamin B6 is a cofactor for more than one hundred enzymatic reactions. The role of CIN has yet only been examined in glioblastoma cell line models derived under serum culture conditions. RESULTS: We found that CIN is highly expressed in cells cultured under non-adherent, serum-free conditions that are thought to better mimic the in vivo situation. Furthermore, the substrates of CIN, p-Ser3-cofilin and active vitamin B6, were significantly reduced as compared to cell lines cultured in serum-containing medium. To further examine its molecular role we stably knocked down the CIN protein with two different shRNA hairpins in the glioblastoma cell lines NCH421k and NCH644. Both cell lines did not show any significant alterations in proliferation but expression of differentiation markers (such as GFAP or TUBB3) was increased in the knockdown cell lines. In addition, colony formation was significantly impaired in NCH644. Of note, in both cell lines CIN knockdown increased active vitamin B6 levels with vitamin B6 being known to be important for S-adenosylmethionine biosynthesis. Nevertheless, global histone and DNA methylation remained unaltered as was chemoresistance towards temozolomide. To further elucidate the role of phosphocofilin in glioblastoma cells we applied inhibitors for ROCK1/2 and LIMK1/2 to our model. LIMK- and ROCK-inhibitor treatment alone was not toxic for glioblastoma cells. However, it had profound, but antagonistic effects in NCH421k and NCH644 under chemotherapy. CONCLUSION: In non-adherent glioblastoma cell lines cultured in serum-free medium, chronophin knockdown induces phenotypic changes, e.g. in colony formation and transcription, but these are highly dependent on the cellular background. The same is true for phenotypes observed after treatment with inhibitors for kinases regulating cofilin phosphorylation (ROCKs and LIMKs). Targeting the cofilin phosphorylation pathway might therefore not be a straightforward therapeutic option in glioblastoma.
Asunto(s)
Neoplasias Encefálicas/patología , Glioblastoma/patología , Fosfoproteínas Fosfatasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Vitamina B 6/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Medio de Cultivo Libre de Suero , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Quinasas Lim/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , ARN Interferente Pequeño/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
The BRAF V600E mutation in exon 15 is considered the disease-defining mutation in hairy cell leukaemia (HCL), but single HCL cases lacking this mutation have been described. In 24 HCL, as well as in 194 various mature B- and T-cell neoplasms, we extended the search for BRAF mutations to exon 11. Two V600E-negative HCL contained novel, potentially functionally relevant mutations in exon 11 (F468C and D449E), while one other HCL was BRAF wild-type in exons 2-17. All non-HCL lymphomas lacked BRAF mutations. We therefore suggest screening of BRAF V600E-negative HCL for alternative exon 11 mutations in the diagnostic setting.
Asunto(s)
Leucemia de Células Pilosas/genética , Linfoma de Células B/genética , Linfoma de Células T/genética , Proteínas Proto-Oncogénicas B-raf/genética , Adulto , Anciano , Anciano de 80 o más Años , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Exones/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas B-raf/química , Análisis de Secuencia de ADNRESUMEN
We studied marginal zone B-cell lymphomas of the gastrointestinal tract including seven small cell lymphomas, eight large cell areas of composite lymphomas and 13 large cell variants using SNP array profiling. We found an increase of genomic complexity with lymphoma progression from small to large cytology, and identified gains of prominent (proto) oncogenes such as REL, BCL11A, ETS1, PTPN1, PTEN and KRAS which were found exclusively in the large cell variants. Copy numbers of ADAM3A, SCAPER and SIRPB1 were varying between the three different modes of presentation, hence suggestive for aberrations associated with progression from small to large cell lymphoma. The number of aberrations was slightly higher in the large cell part of composite lymphomas than in large cell lymphomas, suggesting that clonal selection takes place and that composite lymphomas are in a transition state. To further investigate this, we comparatively analyzed samples of two morphologically different regions of the same small cell tumor with a BIRC3-MALT1 translocation, as well as material acquired at two different time points from one composite lymphoma. We found genomic heterogeneity in both cases, supporting the theory of competing subclones in the evolution and progression of extranodal marginal zone B-cell lymphoma.
Asunto(s)
Evolución Clonal , Neoplasias Gastrointestinales/genética , Linfoma de Células B de la Zona Marginal/genética , Linfoma de Células B Grandes Difuso/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Biomarcadores de Tumor/genética , Caspasas/genética , Femenino , Neoplasias Gastrointestinales/patología , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Linfoma de Células B de la Zona Marginal/patología , Linfoma de Células B Grandes Difuso/patología , Masculino , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/genética , Polimorfismo de Nucleótido Simple , Proto-Oncogenes , Translocación Genética , Ubiquitina-Proteína LigasasRESUMEN
The transcription factor KLF4 may act both as an oncogene and a tumor suppressor in a tissue-depending manner. In T- and pre-B-cell lymphoma, KLF4 was found to act as tumor suppressor. We found the KLF4 promoter methylated in B-cell lymphoma cell lines and in primary cases of B-cell lymphomas, namely, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, and in classic Hodgkin lymphoma (cHL) cases. Promoter hypermethylation was associated with silencing of KLF4 expression. Conditional overexpression of KLF4 in Burkitt lymphoma cell lines moderately retarded proliferation, via cell-cycle arrest in G(0)/G(1). In the cHL cell lines, KLF4 induced massive cell death that could partially be inhibited with Z-VAD.fmk. A quantitative reverse-transcribed polymerase chain reaction array revealed KLF4 target genes, including the proapoptotic gene BAK1. Using an shRNA-mediated knock-down approach, we found that BAK1 is largely responsible for KLF4-induced apoptosis. In addition, we found that KLF4 negatively regulates CXCL10, CD86, and MSC/ABF-1 genes. These genes are specifically up-regulated in HRS cells of cHL and known to be involved in establishing the cHL phenotype. We conclude that epigenetic silencing of KLF4 in B-cell lymphomas and particularly in cHL may favor lymphoma survival by loosening cell-cycle control and protecting from apoptosis.
Asunto(s)
Linfoma de Burkitt/metabolismo , Genes Supresores de Tumor , Enfermedad de Hodgkin/metabolismo , Factores de Transcripción de Tipo Kruppel/fisiología , Linfoma Folicular/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , Apoptosis , Linfocitos B/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Linfoma de Burkitt/genética , Linfoma de Burkitt/patología , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Niño , Preescolar , Metilación de ADN , ADN de Neoplasias/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/patología , Humanos , Factor 4 Similar a Kruppel , Linfoma Folicular/genética , Linfoma Folicular/patología , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Destructora del Antagonista Homólogo bcl-2/antagonistas & inhibidores , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismoRESUMEN
Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is an indolent B-cell lymphoma, which is often localized in the stomach. It is characterized by typical morphology, immunology, cytogenetics and expression profile. The coexistence of a large B-cell lymphoma and a MALT lymphoma in the gastrointestinal tract is defined as a composite lymphoma (ComL) and, as we have previously shown, is almost always the consequence of secondary transformation of MALT lymphoma. Here, we have analyzed a panel of seven MALT lymphomas, seven ComL and thirteen large cell variants of marginal zone B-cell lymphomas (MZBL) using FISH for the detection of rearrangements of IGH, MALT1, BCL6, BCL10 and FOXP1 and immunohistochemistry for Bcl6, Bcl10 and FoxP1. Translocations involving IGH were found in 10/27 lymphomas including two cases with IGH-BCL6 fusion and one with IGH-BCL10 fusion; in 7/10 cases, the translocation partner was not identified. Bcl10 and FoxP1 protein expression was heterogeneous throughout the series. Genetic rearrangements of BCL6 and Bcl6 protein expression were found almost exclusively in the large cell components of the ComL and the large cell extranodal MZBL (p = 0.2093 and p = 0.0261, respectively). These findings suggest Bcl6 as a marker for transformation of MALT lymphoma.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Linfoma de Células B de la Zona Marginal/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Aberraciones Cromosómicas , Proteínas de Unión al ADN/genética , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Linfoma de Células B de la Zona Marginal/patología , Proteínas Proto-Oncogénicas c-bcl-6RESUMEN
Gastrointestinal (g.i.) large cell lymphoma is currently regarded as diffuse large B-cell lymphoma (DLBCL) despite a more favorable clinical outcome compared to other DLBCL. Cluster analyses on a transcriptome signature of NF-κB target genes of 30 g.i. marginal zone B-cell lymphomas (MZBL; 8 g.i. MZBL, 22 large cell MZBL - among them 9 with coexisting small cell component) and 6 DLBCL (3 activated B-cell like (ABC), 3 germinal center-like (GCB)) reveals a distinct pattern. The distinctiveness of large cell MZBL samples is further confirmed by a cohort of 270 available B-cell lymphoma and B-cell in silico profiles. Of the NF-κB genes analyzed, c-REL was overexpressed in g.i. MZBL. c-REL amplification was limited to 6/22 large cell MZBL including the large cell component of 2/9 composite small cell/large cell lymphomas, and c-Rel protein expression was found in the large cell compartment of composite lymphomas. Classification experiments on DLBCL and large cell MZBL profiles support the concept that the large cell MZBL is a distinct type of B-cell lymphoma.