RESUMEN
Rac3 may play an important role in tumor growth but little is known about its expression and mutation in human tumor tissues. We examined the expression of Rac3 using RT-PCR and mutation of the Rac3 gene by DNA sequencing. Overexpression of the Rac3 gene occurred in 19% (5/26) of brain tumors; 3 of 9 (33%) meningiomas, 1 of 11 (9%) astrocytomas and 1 of 6 (17%) pituitary adenomas. Two of the 3 meningiomas with Rac3 overexpression were recurrent meningiomas. The only astrocytoma with Rac3 overexpression was a glioblastoma multiforme. Mutation of the Rac3 gene occurred in 63% (12/19) of brain tumours; 4 of 7 (57.1%) meningiomas, 4 of 5 (80%) pituitary adenomas and 4 of 7 (57.1%) astrocytomas. Except in one astrocytoma, the other four tumors with Rac3 overexpression (3 meningiomas and one pituitary adenoma) did not have Rac3 mutations. Our data is the first report of the frequency of Rac3 overexpression and mutation in human brain tumors. Overexpression may be associated with aggressive tumor behavior. The relationship between Rac3 expression and mutation requires further investigation.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Encefálicas/genética , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica/genética , Mutación/genética , Proteínas de Unión al GTP rac/genética , Astrocitoma/diagnóstico , Astrocitoma/genética , Astrocitoma/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/metabolismo , Cromosomas Humanos Par 17/genética , Análisis Mutacional de ADN , GTP Fosfohidrolasas/metabolismo , Pruebas Genéticas , Humanos , Meningioma/diagnóstico , Meningioma/genética , Meningioma/metabolismo , Invasividad Neoplásica/genética , Neoplasias Hipofisarias/diagnóstico , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/metabolismo , Valor Predictivo de las Pruebas , ARN Mensajero/análisis , ARN Mensajero/genética , Transducción de Señal/genéticaRESUMEN
Wnt regulates developmental and oncogenic processes through its downstream effector, beta-catenin, and a set of other intracellular regulators that are largely conserved among species. E-cadherin was discovered as a protein associated with beta-catenin which plays a crucial role in cell-cell adhesion. To further understand the molecular basis of Wnt signaling pathway and E-cadherin in brain tumorigenesis, the expression of four Wnt genes (Wnt1, Wnt5a, Wnt10b and Wnt13) and E-cadherin were analyzed by reverse transcriptase-polymerase chain reaction. In addition, their downstream effector, beta-catenin, was also investigated. The results showed that the expression of Wnt5a (41/45), Wnt10b (37/45), and Wnt13 (35/45) were found in brain tumors, whereas Wnt1 (6/45) was shown to be less related. Interestingly, E-cadherin was only expressed in a few cases of astrocytoma (2/16), whereas it was expressed in most meningioma (14/15) and pituitary adenoma tumors (12/14). There was no apparent difference of beta-catenin expression profile in brain tumors; however, the sequencing data of beta-catenin showed two mutations on speculative phosphorylation sites, S73F and S23G in astrocytoma. Furthermore, an in vitro functional assay showed that S73F and S23G mutants of beta-catenin did not affect transcriptional activity in TCF-4-leuciferase reporter construct, suggesting that they may need more complex factors to participate in astrocytoma. Taken together, our data suggest that the mutations of beta-catenin together with E-cadherin and Wnt signaling might be involved in brain tumorigenesis.
Asunto(s)
Neoplasias Encefálicas/genética , Cadherinas/genética , Proteínas del Citoesqueleto/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Proteínas de Pez Cebra , Secuencia de Bases , Cartilla de ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Mutagénesis Sitio-Dirigida , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes/metabolismo , Proteínas Wnt , Proteína Wnt1 , beta CateninaRESUMEN
Tcf family transcription factors are the downstream effectors of the Wnt signal transduction pathway that regulates developmental and oncogenetic processes among species. The alternative splicing of consecutive exons in the 3' part of the Tcf-4 gene and an error-prone A9 repeat in exon 17 have recently been examined in several tumors. To further understand the roles played by differential expression and splicing isoforms of human Tcf-4 in brain tumorigenesis, the expression of the 3' part of the Tcf-4 gene (exons 10-17) was analyzed by RT-PCR, nested RT-PCR and DNA sequencing. The results showed that at least 13 of the Tcf-4 alternative splicing isoforms were found and most of them were overexpressed in various brain tumors. L3 isoform was particularly dominant in metastasis. Several novel splicing isoforms were identified. One that contains different combinations of exon 16 (10-11-12-16-17, S5) was found in normal brain and pituitary adenoma but not in astrocytoma, meningioma or metastasis, whereas the other that contains part of intron 16, designated exon-16', was found in metastasis. Overall 23% of sequencing analysis in brain tumors exhibited frameshift mutations in an A9 repeat region of exon 17. These mutants exhibited 2-base or 1-base deletion (A7, A8) and 1-base insertion (A10). Nonetheless, in vitro functional assay showed that these mutants did not affect the transactivity of Tcf-4 comparing to wild-type Tcf-4. Collectively, our data suggest that a large number of alternative splicing isoforms may together with variable mutations of A9 repeat region maintain balanced pools of Tcf-4 isoforms during brain tumorigenesis.
Asunto(s)
Adenoma/genética , Adenoma/patología , Astrocitoma/genética , Astrocitoma/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Proteínas de Unión al ADN/biosíntesis , Perfilación de la Expresión Génica , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/patología , Factores de Transcripción/biosíntesis , Astrocitoma/secundario , Neoplasias Encefálicas/secundario , Análisis Mutacional de ADN , Femenino , Secuencias Hélice-Asa-Hélice , Humanos , Masculino , Isoformas de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transducción de Señal , Factores de Transcripción TCF , Proteína 2 Similar al Factor de Transcripción 7RESUMEN
The human dynamin-like protein, HdynIV, has recently been cloned and shown to be involved in the formation and trafficking of coated vesicles. In particular, one of the HdynIV variant overexpressions has been suggested to contribute to the pathogenesis of brain tumors. In this paper, we report on the genomic organization of the human HdynIV gene. The gene was found to correspond to 20 exons of genomic sequence on human chromosome 12, distributed over 64kb of genomic DNA. The two exons, numbers 15 and 16, are subjected to differential splicing, generating four different transcripts of a perfect match to our recent report on the four different spliced HdynIV variants [DNA Cell Biol. 19 (2000) 189]. We have also characterized the 5(') regulatory region of the HdynIV gene in order to understand the molecular mechanisms regulating its expression. The transcriptional initiation site was identified by 5(')-RACE. The 5(')-flanking sequence of the HdynIV gene contains three GC boxes that concatenate Ap2- and Sp1-binding motifs, but that does not contain either the TATA or CAAT consensus sequence. A region between -140 and +92 contributed to high promoter activity. Deletion analysis demonstrated that the minimal promoter activity required the region of -110 to -100. Electrophoretic mobility shift assay demonstrated that a putative transcriptional factor bound to the region of -119 to -90. Site-directed mutagenesis analysis of this region revealed that nucleotides at -108 to -100 were essential for transactivation mediated by this transcriptional factor. In conclusion, we have characterized the minimal HdynIV promoter and shown that CTCCCAGCA (-108 to -100) sequence may act as a novel transcriptional element for regulating HdynIV gene expression.