RESUMEN

Female meiotic divisions in higher organisms are asymmetric and lead to the formation of a large oocyte and small polar bodies. These asymmetric divisions are due to eccentric spindle positioning which, in the mouse, requires actin filaments. Recently Formin-2, a straight actin filaments nucleator, has been proposed to control spindle positioning, chromosome segregation as well as first polar body extrusion in mouse oocytes. We reexamine here the possible role of Formin-2 during mouse meiotic maturation by live videomicroscopy. We show that Formin-2 controls first meiotic spindle migration to the cortex but not chromosome congression or segregation. We also show that the lack of first polar body extrusion in fmn2(-/-) oocytes is not due to a lack of cortical differentiation or central spindle formation but to a defect in the late steps of cytokinesis. Indeed, Survivin, a component of the passenger protein complex, is correctly localized on the central spindle at anaphase in fmn2(-/-) oocytes. We show here that attempts of cytokinesis in these oocytes abort due to phospho-myosin II mislocalization.

Asunto(s)

Citocinesis/fisiología , Proteínas del Tejido Nervioso/fisiología , Oocitos/citología , Huso Acromático , Animales , Cromosomas , Ratones

RESUMEN

The tumor suppressor gene, p53, is rarely mutated in neuroblastomas (NB) at the time of diagnosis, but its dysfunction could result from a nonfunctional conformation or cytoplasmic sequestration of the wild-type p53 protein. However, p53 mutation, when it occurs, is found in NB tumors with drug resistance acquired over the course of chemotherapy. As yet, no study has been devoted to the function of the specific p53 mutants identified in NB cells. This study includes characterization and functional analysis of p53 expressed in eight cell lines: three wild-type cell lines and five cell lines harboring mutations. We identified two transcription-inactive p53 variants truncated in the C-terminus, one of which corresponded to the p53beta isoform recently identified in normal tissue by Bourdon et al. [J. C. Bourdon, K. Fernandes, F. Murray-Zmijewski, G. Liu, A. Diot, D. P. Xirodimas, M. K. Saville and D. P. Lane (2005) Genes Dev., 19, 2122-2137]. Our results show, for the first time, that the p53beta isoform is the only p53 species to be endogenously expressed in the human NB cell line SK-N-AS, suggesting that the C-terminus truncated p53 isoforms may play an important role in NB tumor development.

Asunto(s)

Neuroblastoma/genética , Eliminación de Secuencia , Proteína p53 Supresora de Tumor/genética , Secuencia de Bases , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Dosificación de Gen , Expresión Génica , Humanos , Datos de Secuencia Molecular , Neuroblastoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Levaduras/genética

RESUMEN

The present study aims to investigate the role of p73 in response to cisplatin treatment in p53 wild-type neuroblastoma SH-SY5Y cells. Results showed that cisplatin induced a dose-dependent up-regulation of p53, p73, and a number of p53-responsive genes. Interestingly, endogenous Deltaexon2p73-expression was down-regulated by cisplatin treatment. Neither p21 nor GADD45 induction was observed in p53-deficient Lan-1 cells, although endogenous TAp73 expression was markedly induced. In the presence of cisplatin, exogenous TAp73 overexpression in SH-SY5Y cells induced p21 up-regulation without altering the apoptotic sub-G1 cell population. Moreover, siRNA-mediated suppression of TAp73 expression did not alter the sub-G1 population. Collectively, our results suggest that wt-p53 SH-SY5Y cells respond to cisplatin by inducing p73 isoform regulation and sustaining p53-dependent apoptosis that is independent of TAp73alpha.

Asunto(s)

Antineoplásicos/farmacología , Cisplatino/farmacología , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Empalme Alternativo , Apoptosis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Relación Dosis-Respuesta a Droga , Humanos , Neuroblastoma , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Estabilidad del ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Factores de Tiempo , Transfección , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética

RESUMEN

The p53 gene and its homologue p73 are rarely mutated in neuroblastoma. In recent studies, we showed that overexpression of DeltaNp73alpha, an isoform lacking the N-terminal transactivation (TA) domain, surprisingly induces p53 protein accumulation in the wild-type (wt) p53 human neuroblastoma line SH-SY5Y. As can be expected owing to its dominant-negative effect, DeltaNp73alpha inhibits Waf1/p21 gene expression, but equally importantly, it upregulates BTG2TIS21/PC3, another p53 target gene. This effect is not observed in neuroblastoma cells that express a mutated p53. To better understand the DeltaNp73-mediated transactivation of the BTG2TIS21/PC3 gene we performed luciferase assays with two reporter plasmids harboring long and short BTG2 promoter sequences in three human neuroblastoma cell lines and one breast cancer cell line. Our results demonstrate that BTG2TIS21/PC3 transactivation by DeltaNp73alpha depends on both p53 status (as it is not observed in a p53-/- neuroblastoma cell line) and cellular context (as it occurs in a p53+/+ neuroblastoma cell line but not in a p53+/+ breast tumor cell line). The fact that DeltaNp73alpha may either inhibit or stimulate wt-p53 transcriptional activity, depending on both the p53 target gene and the cellular context, was confirmed by real-time quantitative PCR. Moreover, transactivation of the BTG2TIS21/PC3 promoter requires a complete DeltaNp73alpha C-terminus sequence as it is not observed with DeltaNp73beta, which lacks most of the C-terminal domain. We have previously shown that DeltaNp73alpha is the only p73 isoform expressed in undifferentiated neuroblastoma tumors. In light of all these findings, we propose that DeltaNp73alpha not only acts as an inhibitor of p53/TAp73 functions in neuroblastoma tumors, but also cooperates with wt-p53 in playing a physiological role through the activation of BTG2TIS21/PC3 gene expression.

Asunto(s)

Proteínas de Unión al ADN/metabolismo , Proteínas Inmediatas-Precoces/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Proteínas Nucleares/metabolismo , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Adenoviridae/genética , Apoptosis , Western Blotting , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Genes Reporteros , Genes Supresores de Tumor , Humanos , Luciferasas/metabolismo , Plásmidos/metabolismo , Isoformas de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Proteínas Supresoras de Tumor , Regulación hacia Arriba

RESUMEN

Homologies in sequence and gene organization of p53 and their relatives, p73 and p63, suggest similar biological functions. However differences exist between the p53 family members. Indeed in human tumors p53 is often mutated while p63 and p73 are very rarely mutated. In addition, in contrast to p53 which is transcribed in a unique mRNA species spanning all gene exons, each homologue expresses two types of isoforms: some with transactivation domain (TAD) showing tumor suppressive properties, the others deprived of TAD, with oncogenic properties. If p53 responds to immediate genotoxic stress, its homologues participate to the cell homeostasis of specific tissues along their development and differentiation, neuronal tissue for p73, epithelial for p63. However a collaboration between the three p53 family members has been shown to occur in response to cell genotoxic damages. Neuroblastic tumors characterized by a large spectrum of neuronal differentiation constitute a good model to study relationship between p73 and p53 as well as the regulation of their respective expression.

Asunto(s)

Transformación Celular Neoplásica , Proteínas de Unión al ADN , Genes p53 , Predisposición Genética a la Enfermedad , Neuroblastoma/genética , Proteínas Nucleares , Diferenciación Celular , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Neuronas/fisiología , ARN Mensajero , Proteína Tumoral p73 , Proteínas Supresoras de Tumor