RESUMEN
Mxi1 is a Mad family member that plays a role in cell proliferation and differentiation. To test the role of Mxi1 on tumorigenesis of glioma cells we transfected a CMV-driven MXI1 cDNA in U87 human glioblastoma cells. Two clones were isolated expressing MXI1 levels 18- and 3.5-fold higher than wild-type U87 cells (clone U87.Mxi1.14 and U87.Mxi1.22, respectively). In vivo, U87.Mxi1.14 cells were not tumorigenic in nude mice and delayed development of tumours was observed with U87.Mxi1.22 cells. In vitro, the proliferation rate was partially and strongly inhibited in U87.Mxi1.22 and U87.Mxi1.14 cells respectively. The cell cycle analysis revealed a relevant accumulation of U87.Mxi1.14 cells in the G(2)/M phase. Interestingly, the expression of cyclin B1 was inhibited to about 60% in U87.Mxi1.14 cells. This inhibition occurs at the transcriptional level and depends, at least in part, on the E-box present on the cyclin B1 promoter. Consistent with this, the endogenous Mxi1 binds this E-box in vitro. Thus, our findings indicate that Mxi1 can act as a tumour suppressor in human glioblastomas through a molecular mechanism involving the transcriptional down-regulation of cyclin B1 gene expression.
Asunto(s)
Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/fisiología , Ciclina B/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Factores de Transcripción/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Ciclo Celular/fisiología , Ciclina B1 , Proteínas de Unión al ADN/genética , Fase G2 , Genes Reporteros , Glioma , Humanos , Cinética , Mitosis , Proteínas Recombinantes/farmacología , Factores de Tiempo , Factores de Transcripción/genética , Transcripción Genética , Transfección , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
In mammalian cells, the expression level of the cyclin B1 gene plays a critical role in the progression through mitosis. Here we demonstrate that the transcriptional activity of the human cyclin B1 promoter, as well as the rate of gene transcription, is high during mitosis. Indeed, the cyclin B1 promoter maintains an open chromatin configuration at the mitotic stage. Consistent with this, we show that the cyclin B1 promoter is occupied and bound to NF-Y during mitosis in vivo. Our results provide the first example of RNA polymerase II-dependent transcription during mitosis in mammalian cells.
Asunto(s)
Ciclina B/biosíntesis , Ciclina B/genética , Mitosis , Transcripción Genética , Northern Blotting , Factor de Unión a CCAAT/metabolismo , Cromatina/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Ciclina B1 , ADN Complementario/metabolismo , Fase G2 , Células HeLa , Humanos , Modelos Genéticos , Pruebas de Precipitina , Regiones Promotoras Genéticas , Unión Proteica , Conformación ProteicaRESUMEN
During normal cell cycles, the function of mitotic cyclin-cdk1 complexes, as well as of cdc25C phosphatase, is required for G2 phase progression. Accordingly, the G2 arrest induced by DNA damage is associated with a down-regulation of mitotic cyclins, cdk1, and cdc25C phosphatase expression. We found that the promoter activity of these genes is repressed in the G2 arrest induced by DNA damage. We asked whether the CCAAT-binding NF-Y modulates mitotic cyclins, cdk1, and cdc25C gene transcription during this type of G2 arrest. In our experimental conditions, the integrity of the CCAAT boxes of cyclin B1, cyclin B2, and cdc25C promoters, as well as the presence of a functional NF-Y complex, is strictly required for the transcriptional inhibition of these promoters. Furthermore, a dominant-negative p53 protein, impairing doxorubicin-induced G2 arrest, prevents transcriptional down-regulation of the mitotic cyclins, cdk1, and cdc25C genes. We conclude that, as already demonstrated for cdk1, NF-Y mediates the transcriptional inhibition of the mitotic cyclins and the cdc25C genes during p53-dependent G2 arrest induced by DNA damage. These data suggest a transcriptional regulatory role of NF-Y in the G2 checkpoint after DNA damage.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B/genética , Fase G2/fisiología , Regiones Promotoras Genéticas/genética , Fosfatasas cdc25/genética , Factor de Unión a CCAAT/genética , Células Cultivadas , Ciclina B1 , Daño del ADN , Regulación hacia Abajo , Doxorrubicina/farmacología , Mitosis , Músculo Esquelético/citología , Transcripción Genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The observation that cyclin B1 protein and mRNAs are down-regulated in terminally differentiated (TD) C2C12 cells, suggested us to investigate the transcriptional regulation of the cyclin B1 gene in these cells. Transfections of cyclin B1 promoter constructs indicate that two CCAAT boxes support cyclin B1 promoter activity in proliferating cells. EMSAs demonstrate that both CCAAT boxes are recognized by the trimeric NF-Y complex in proliferating but not in TD cells. Transfecting a dominant-negative mutant of NF-YA we provide evidence that NF-Y is required for maximal promoter activity. Addition of recombinant NF-YA to TD C2C12 nuclear extracts restores binding activity in vitro, thus indicating that the loss of NF-YA in TD cells is responsible for the lack of the NF-Y binding to the CCAAT boxes. Consistent with this, we found that the NF-YA protein is absent in TD C2C12 cells. In conclusion, our data demonstrate that NF-Y is required for cyclin B1 promoter activity. We also demonstrate that cyclin B1 expression is regulated at the transcriptional level in TD C2C12 cells and that the switch-off of cyclin B1 promoter activity in differentiated cells depends upon the loss of a functional NF-Y complex. In particular the loss of NF-YA protein is most likely responsible for its inactivation.
Asunto(s)
Factor de Unión a CCAAT , Ciclina B/genética , Proteínas de Unión al ADN/metabolismo , Músculo Esquelético/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT , Diferenciación Celular/fisiología , División Celular , Células Cultivadas/metabolismo , Ciclina B/metabolismo , Ciclina B1 , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/fisiología , Regulación de la Expresión Génica , Ratones , Músculo Esquelético/citología , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Medullary thyroid carcinoma (MTC) is a rare human tumor affecting the calcitonin-secreting c-cells of the thyroid. Here we report that two independent strains of transgenic mice expressing a Polyomavirus (Py) truncated middle-T antigen (deltaMT), consisting of the amino-terminal 304 amino acids, and the full length Py small-T antigen, developed multifocal bilateral MTCs with 100% penetrance. Occasionally one strain also developed mammary and bone tumors. Furthermore, offspring from both transgenic lines displayed pronounced waviness of the whiskers and fur, previously associated with defective epidermal growth factor receptor signaling. Transgene transcription, driven by the homologous early promoter/enhancer, and the corresponding translation products were detected in tumors and in many other organs which did not develop pathologies. The subcellular distribution of deltaMT and its interactions with the adapter proteins of the SHC family have also been analysed. Our study describes a novel murine model of MTC and provides evidence that the N-terminal 304 amino acid fragment of Py middle-T antigen, possibly in co-operation with small-T antigen, acts as a potent oncogene in c-cells of the thyroid.
Asunto(s)
Antígenos Transformadores de Poliomavirus/fisiología , Carcinoma Medular/genética , Neoplasias de la Tiroides/genética , Animales , Antígenos Transformadores de Poliomavirus/química , Antígenos Transformadores de Poliomavirus/genética , Sitios de Unión , Carcinoma Medular/patología , Receptores ErbB/fisiología , Regulación de la Expresión Génica , Cabello/anomalías , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Neoplasias Primarias Múltiples/genética , Especificidad de Órganos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Eliminación de Secuencia , Neoplasias de la Tiroides/patología , Transgenes , Vibrisas/anomalíasRESUMEN
Cyclin B2 is a regulator of p34cdc2 kinase, involved in G2/M progression of the cell cycle, whose gene is strictly regulated at the transcriptional level in cycling cells. The mouse promoter was cloned and three conserved CCAAT boxes were found. In this study, we analysed the mechanisms leading to activation of the cyclin B2 CCAAT boxes: a combination of (i) genomic footprinting, (ii) transfections with single, double and triple mutants, (iii) EMSAs with nuclear extracts, antibodies and NF-Y recombinant proteins and (iv) transfections with an NF-YA dominant negative mutant established the positive role of the three CCAAT sequences and proved that NF-Y plays a crucial role in their activation. NF-Y, an ubiquitous trimer containing histone fold subunits, activates several other promoters regulated during the cell cycle. To analyse the levels of NF-Y subunits in the different phases of the cycle, we separated MEL cells by elutriation, obtaining fractions >80% pure. The mRNA and protein levels of the histone-fold containing NF-YB and NF-YC were invariant, whereas the NF-YA protein, but not its mRNA, was maximal in mid-S and decreased in G2/M. EMSA confirmed that the CCAAT-binding activity followed the amount of NF-YA, indicating that this subunit is limiting within the NF-Y complex, and suggesting that post-transcriptional mechanisms regulate NF-YA levels. Our results support a model whereby fine tuning of this activator is important for phase-specific transcription of CCAAT-containing promoters.
Asunto(s)
Ciclina B/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT , Ciclo Celular , Línea Celular , Ciclina B2 , Huella de ADN , ADN Complementario , Ratones , Datos de Secuencia MolecularRESUMEN
We recently reported that rhabdomyosarcoma cell lines express and secrete interleukin 15 (IL-15), a tightly regulated cytokine with IL-2-like activity. To test whether the p53-impaired function that is frequently found in this tumour type could play a role in the IL-15 production, wild-type p53 gene was transduced in the human rhabdomyosarcoma cell line RD (which harbours a mutated p53 gene), and its effect on proliferation and expression of IL-15 was studied. Arrest of proliferation was induced by wild-type p53; increased proportions of G1-arrested cells and of apoptotic cells were observed. A marked down-modulation of IL-15 expression, at both the mRNA and protein level, was found in p53-transduced cells. Because a direct effect of IL-15 on normal muscle cells has been reported, the presence of IL-15 membrane receptors was studied by cytofluorometric analysis. Rhabdomyosarcoma cells showed IL-15 membrane receptors, which are down-modulated by wild-type p53 transfected gene. In conclusion, wild-type p53 transduction in human rhabdomyosarcoma cells induces the down-modulation of both IL-15 production and IL-15 receptor expression.
Asunto(s)
Genes p53 , Interleucina-15/metabolismo , Receptores de Interleucina-2/metabolismo , Rabdomiosarcoma/metabolismo , División Celular/genética , Linaje de la Célula , Regulación hacia Abajo , Humanos , Receptores de Interleucina-15 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rabdomiosarcoma/patología , Transcripción Genética , Transducción Genética , Transfección , Células Tumorales CultivadasRESUMEN
Cyclin Bl plays an important role in cell proliferation. Its expression is tightly regulated at the mRNA and protein levels during the cell cycle and is found to be deregulated in various malignancies. To enlighten the signalling pathways which lead to the cell cycle dependent expression of the cyclin B1 gene, we studied its transcriptional regulation in quiescent and proliferating NIH3T3 cells. We previously showed that the transcriptional activity of the cyclin B1 promoter decreases in quiescent cells. Here, we map a quiescence-responsive element of the human cyclin B1 promoter to an E-box sequence, CACGTG, which spans positions -124/-119. Nuclear proteins protect this sequence in a DNase I digestion assay and bind, in electromobility shift assays, an oligonucleotide spanning positions -133/-110. Max-specific antibodies block the DNA-binding activity of protein complexes to this probe. A mutation in the E-box core sequence abolishes the decrease in transcription that occurs during quiescence. Finally, we find that over-expression of Max protein in proliferating cells specifically inhibits cyclin B1 promoter activity through this E-box. Moreover, Max over-expression in proliferating NIH3T3 cells leads to down-regulation of the endogenous cyclin B1 protein. In conclusion, these data support a model whereby E-box-binding proteins mediate the decrease in the transcriptional activity of the cyclin B1 promoter observed in quiescent cells and suggest that Max contributes to this response.
Asunto(s)
Ciclina B , Ciclinas/biosíntesis , Ciclinas/genética , Secuencias Hélice-Asa-Hélice , Regiones Promotoras Genéticas , Factores de Transcripción , Transcripción Genética , Células 3T3/citología , Células 3T3/metabolismo , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , División Celular/fisiología , Ciclina B1 , Proteínas de Unión al ADN/metabolismo , Humanos , Ratones , Mutación , Proteínas Nucleares/metabolismo , Unión ProteicaRESUMEN
As a step toward defining the signals important for the regulation of cyclin B1 expression, we cloned, sequenced, and partially characterized a 1012-bp genomic fragment encompassing the human cyclin B1 promoter. By transient expression experiments, we found that promoter activity resides within a -150/+182-bp DNA fragment. The activity of this promoter fragment was high in asynchronous NIH-3T3 cells, but dramatically decreased in quiescent cells. Time-course experiments, using stable transfectants expressing the CAT gene under the control of this fragment, were performed after releasing the cells from serum starvation. The results showed that the promoter becomes active at the end of the S phase and its activity increases during the cell cycle. Similar experiments performed with a shorter promoter region (-58/+182) showed that this 5' deletion mutant is active throughout the cell cycle. In good agreement with promoter activity, Northern analysis indicated that the endogenous gene is negatively regulated in quiescent murine NIH-3T3 cells. The data presented here demonstrate that in NIH-3T3 cells the cyclin B1 promoter is growth regulated, and important regulatory elements must exist in the region spanning -150 to -58 bp.
Asunto(s)
Ciclinas/genética , Regulación de la Expresión Génica/fisiología , Regiones Promotoras Genéticas/genética , Células 3T3/citología , Animales , Secuencia de Bases , Ciclo Celular , División Celular , Humanos , Ratones , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Análisis de Secuencia de ADN , Eliminación de Secuencia/fisiología , Transcripción Genética/genéticaRESUMEN
We report the first evidence that differential transcriptional regulation of human chromogranin A (CHGA) gene expression occurs during in vitro treatment of tumorigenic neuroblastoma (NB) cells with retinoic acid (5 microM) and/or dibutyryl-cAMP (1 mM). The CHGA gene encodes a tissue specific protein restricted to cells of the diffuse neuroendocrine system, but also widely expressed among NB tumours. We previously reported that CHGA as well as other neuroendocrine markers are modulated during NB differentiation in vitro. To investigate, at the molecular level, the mechanisms leading to NB tumour cell differentiation during the treatment with biologically active compounds, we sequenced and functionally characterised 2169 bp of a genomic DNA clone encompassing the 5' flanking region of the human CHGA gene. Computer-assisted analysis of the sequence revealed the presence of a cAMP responsive element at positions -56 to -49, and Sp1 binding sites at positions -181 to -176 and -216 to -210. Two novel 9 bp motifs, located at position -462 to -454 and -91 to -83 of the CHGA promoter were identified in the regulatory regions of two other neuroendocrine genes encoding for tyrosine hydroxylase and neuropeptide Y. In addition, in the first 1000 bp of the untranslated 5' region, we found the presence of several putative DNA binding sites of bHLH molecules, a protein family regulating tissue specific differentiation. Transient transfection experiments of chloramphenicol acetyltransferase (CAT) deletion constructs, showed the presence of an active promoter within the first 455 bp upstream from the start site. This region conferred tissue specific expression to a CAT reporter gene. In addition, the transcriptional activity of this fragment was modulated during the induction of differentiation of NB cells treated by retinoic acid and/or dibutyryl-cAMP. These observations provide preliminary data regarding CHGA transcriptional regulation in NB cells, and indicate that retinoic acid and cAMP activate distinct, apparently competitive, transcriptional pathways during NB cell differentiation. The molecular characterisation of the mechanisms regulating CHGA expression in tumour and normal neuroendocrine tissue could lead to the identification of novel molecules potentially relevant for future gene therapy of NB tumours.