RESUMO
Desmoplastic small round cell tumor (DSRCT) is a primitive sarcoma with a consistent cytogenetic abnormality, t(11;22)(p13;q12). This chromosomal translocation generates a chimeric transcript that is formed by fusion of the 5' region of the Ewing's sarcoma gene, EWS, with the 3' DNA-binding segment of WT1, the Wilms' tumor suppressor gene. We collected 14 DSRCT tumor samples and examined the hybrid transcripts. We identified: (a) combinatorial heterogeneity of EWS exons fused to WT1 including use of EWS exons 7, 8, and 9; (b) subpopulations of variant transcripts in 6 of 14 tumors characterized by aberrant splicing resulting in loss of EWS exon 6 or WT1 exon 9; (c) multiple cDNA products with large internal deletions; and (d) insertion of small stretches of heterologous DNA at the fusion site or exon splice region in transcripts from two tumors. Most of the splice variants were in-frame, and in vitro translated fusion proteins with intact DNA-binding motifs formed complexes with a WT1 response element in gel mobility assays. Each of the chimeric proteins retains the ability to bind to the GC and TC elements of the early transcription factor EGR-1 as well as WT1 consensus sequences. We present evidence that various EWS-WT1 proteins up-regulated EGR-1 promoter activity and that this up-regulation is specifically dependent upon the absence of the exon 9 KTS domain of WT1. The molecular diversity and functionality exhibited by these fusion transcripts may have significant biological implications for their transactivating and tumorigenic potential.
Assuntos
Neoplasias Abdominais/genética , Carcinoma de Células Pequenas/genética , Proteínas Imediatamente Precoces , Proteínas de Fusão Oncogênica/fisiologia , Adolescente , Adulto , Sítios de Ligação , Criança , Proteínas Quimerinas/genética , DNA Complementar/genética , DNA Complementar/metabolismo , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Feminino , Genes do Tumor de Wilms/genética , Humanos , Masculino , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Ewing/genética , Fatores de Transcrição/genética , Ativação Transcricional/fisiologia , Proteínas WT1 , Dedos de Zinco/fisiologiaRESUMO
L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to unique carbohydrate ligands, sulfated sialyl Lewis(x), which are expressed on high endothelial venules (HEV) in secondary lymphoid organs. The nature of the sulfotransferase(s) that contribute to sulfation of such L-selectin counterreceptors has been uncertain. We herein describe a novel L-selectin ligand sulfotransferase, termed LSST, that directs the synthesis of the 6-sulfo sialyl Lewis(x) on L-selectin counterreceptors CD34, GlyCAM-1, and MAdCAM-1. LSST is predominantly expressed in HEV and exhibits striking catalytic preference for core 2-branched mucin-type O-glycans as found in natural L-selectin counterreceptors. LSST enhances L-selectin-mediated adhesion under shear compared to nonsulfated controls. LSST therefore corresponds to an HEV-specific sulfotransferase that contributes to the biosynthesis of L-selectin ligands required for lymphocyte homing.