IDENTIFICATION OF TARGET GENES FOR ETS-1 GENE PRODUCT

ETS-1 基因产物的靶基因的鉴定

• 批准号: 3838493
• 负责人: T S PAPAS
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3838493
• 关键词: binding proteins; gel electrophoresis; gene expression; molecular cloning; oncogenes; protein purification; stainings; transcription factor; transfection; western blottings

The ETS1 gene encodes a transcription factor, and binds to purine-rich sequences in the cellular and viral promoters and enhancers. To identify new target genes, ETS1 proteins are expressed in DLD-1 cells and change in the levels of proteins is examined by two-dimensional gel electrophoresis analysis of total cellular proteins. The human colon cancer cell line, DLD-1, shows no detectable expression of ETS1. By co-transfection with human ETS1 and pSV2neo expression vectors into DLD-1 cells, two independent clones, S1 and S7, expressing full- length ETS1 protein were selected. Clone S7 expresses ETS1 protein at much higher levels than clone S1. Total cellular proteins were analyzed by two-dimensional gel analysis and proteins were visualized by silver staining. The level of p54.5-6.6 (designated by molecular weight x 10 -3 and pI) increased in both S1 and S7 clones compared with three independent DLD-1 subclones not expressing ETS1 protein. The expression level of p54.5-6.6 seemed to parallel that of the ETS1 protein. The p54.5-6.6 was different from ETS1 protein by Western blot analysis. These results suggest that p54.5-6.6 is a candidate target gene product for ETS1 protein. Further identification of the p54.5-6.6 protein is in progress. Clone S7, expressing ETS1 protein abundantly, shows reduced tumorigenicity in colony formation assays in soft agar and in the nude mice assay. To confirm that reduced tumorigenicity is due to high expression of ETS1 in DLD-1 cells, other clones are being selected by independent transfection.

ETS 1基因编码一种转录因子，并与富含嘌呤的 细胞和病毒启动子和增强子中的序列。 以识别 新的靶基因，ETS 1蛋白在DLD-1细胞中表达，并在DLD-1细胞中改变。 通过双向凝胶电泳检测蛋白质水平 总细胞蛋白的分析。 人结肠癌细胞系DLD-1没有显示出可检测到的 ETS 1. 通过与人ETS 1和pSV 2neo表达载体共转染 在DLD-1细胞中，两个独立的克隆，S1和S7，表达完整的 长度ETS 1蛋白。 克隆S7表达ETS 1蛋白， 远远高于克隆S1。 通过二维凝胶分析分析总细胞蛋白， 通过银染色使蛋白质可视化。 p54.5-6.6水平 （以分子量× 10 - 3和pI表示）在S1和S2中均增加， S7克隆与三个独立的DLD-1亚克隆相比， ETS 1蛋白。 p54.5-6.6的表达水平似乎与 ETS 1蛋白质 p54.5-6.6蛋白与ETS 1蛋白有明显差异， 蛋白质印迹分析。 这些结果表明，p54.5-6.6是一个 ETS 1蛋白的候选靶基因产物。 进一步查明 p54.5-6.6蛋白正在进行中。 大量表达ETS 1蛋白的克隆S7显示出降低的致瘤性 在软琼脂中的集落形成试验和裸鼠试验中。 到 证实了降低的致瘤性是由于ETS 1的高表达， DLD-1细胞，其他克隆通过独立转染进行选择。