RESUMO
OBJECTIVES: To determine the expression level of the Wnt components-WIF1 (Wnt inhibitory factor 1), WNT1, and beta-catenin-in salivary gland tumor cells and to investigate the mechanisms that contribute to activation of the Wnt pathway in human salivary gland tumors. DESIGN: The expression of WIF1, WNT1, and beta-catenin in salivary gland normal tissue and tumor cell lines was analyzed by reverse transcription-polymerase chain reaction and Western blot analysis. A relationship between the expression of distinct genes was determined by Pearson correlation. The presence of rearrangements involving WIF1 was evaluated by reverse transcription-polymerase chain reaction analysis. SUBJECTS: Samples were obtained from 6 normal salivary glands and 10 cell lines established from primary benign and malignant salivary gland tumors. RESULTS: The expression of WIF1 was high in normal salivary gland tissue but was significantly down-regulated in all salivary gland tumor cell lines analyzed (P < .001). The WIF1 rearrangements were recurrent but rare in salivary gland tumors. Expression of WNT1 protein was undetectable in normal tissue but readily detectable by Western blot analysis in all salivary gland tumor cell lines. beta-Catenin messenger RNA expression was significantly up-regulated in salivary gland tumor cells. A positive linear correlation between beta-catenin and PLAG1 (pleomorphic adenoma gene 1) gene expression was observed. CONCLUSIONS: This is the first report (to our knowledge) showing down-regulation of an antagonist of the Wnt pathway, WIF1, and up-regulation of a Wnt agonist, WNT1, in salivary gland tumor cells. This dysregulation of WNT1 and WIF1 expression, coupled with the observed increase in beta-catenin transcription, may consequently promote salivary gland oncogenesis. Our data support the study of the Wnt pathway as a putative therapeutic target for salivary gland cancer.
Assuntos
Neoplasias das Glândulas Salivares/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Western Blotting , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Humanos , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Regulação para Cima , beta Catenina/metabolismoRESUMO
Nucleotide excision repair (NER) and RNA polymerase II (Pol II) transcription are essential cellular processes which are intimately intertwined. They share an indispensable multiprotein complex, TFIIH, and impairments in either process can impact the efficiency of the other. Like TFIIH, MMS19 is required for NER and Pol II transcription, but its precise role in each process is unknown. We showed previously that the human MMS19 gene originates multiple splice variants, some of which may encode distinct MMS19 protein isoforms. Here we characterize a novel MMS19 transcript and demonstrate for the first time that MMS19 splice variants are conserved across species and are functionally distinct. Expression of human MMS19 splice variants in mms19-deleted yeast cells produced unique patterns of thermosensitivity and ultraviolet radiation-sensitivity that point to three MMS19 structural domains with distinct in vivo functions. MMS19 polypeptides lacking domain A are able to fulfill the role of full-length MMS19 in NER but not in transcription. MMS19 polypeptides lacking part of domain B are efficient in transcription but not in NER. MMS19 polypeptides lacking domain C (HEAT repeats) are unable to fulfill either function. Our data suggest that the MMS19 HEAT repeat domain is essential for MMS19 function in NER and transcription, while domains A and B, within MMS19 N-terminus, modulate the balance between DNA repair and transcription. Our results highlight the functional significance of MMS19 transcripts and the possible contribution of MMS19 isoforms to regulate the switch between NER and transcription. Furthermore, our work associates for the first time specific protein domains with MMS19's role in NER and transcription.
Assuntos
Reparo do DNA/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética , Processamento Alternativo/genética , Análise de Variância , Northern Blotting , Western Blotting , Sequência Conservada/genética , Primers do DNA , Teste de Complementação Genética , Humanos , Estrutura Terciária de Proteína/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos da radiação , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
Bright (B-cell regulator of immunoglobulin heavy chain transcription) binding to immunoglobulin heavy chain loci after B-cell activation is associated with increased heavy chain transcription. Our earlier reports demonstrated that Bright coimmunoprecipitates with Bruton's tyrosine kinase (Btk) and that these proteins associate in a DNA-binding complex in primary B cells. B cells from immunodeficient mice with a mutation in Btk failed to produce stable Bright DNA-binding complexes. In order to determine if Btk is important for Bright function, a transcription activation assay was established and analyzed using real-time PCR technology. Cells lacking both Bright and Btk were transfected with Bright and/or Btk along with an immunoglobulin heavy chain reporter construct. Immunoglobulin gene transcription was enhanced when Bright and Btk were coexpressed. In contrast, neither Bright nor Btk alone led to activation of heavy chain transcription. Furthermore, Bright function required both Btk kinase activity and sequences within the pleckstrin homology domain of Btk. Bright was not appreciably phosphorylated by Btk; however, a third tyrosine-phosphorylated protein coprecipitated with Bright. Thus, the ability of Bright to enhance immunoglobulin transcription critically requires functional Btk.
