IDENTIFICATION OF TARGET GENES FOR ETS-1 GENE PRODUCT

ETS1基因编码一种转录因子，并与富含嘌呤的物质结合 细胞和病毒启动子和增强子中的序列。要确定 新的靶基因ETS1蛋白在DLD-1细胞中表达，并在 用双向凝胶电泳法检测蛋白质水平。 细胞总蛋白的分析。 人结肠癌细胞系DLD-1没有检测到表达 ETS1。通过与人ETS1和pSV2neo表达载体共转染 将两个相互独立的克隆S1和S7导入DLD-1细胞，表达完整的 选择长度较长的ETS1蛋白。克隆S7表达ETS1蛋白 比克隆人S1的水平高得多。 细胞总蛋白的二维凝胶分析和 银染可见蛋白质的表达。P54.5-6.6的水平 (用分子量x 10-3和pI表示)在S1和 S7克隆与3个独立的不表达DLD-1亚克隆的比较 ETS1蛋白。P54.5-6.6的表达水平似乎与之平行 ETS1蛋白。P54.5-6.6不同于ETS1蛋白 蛋白质印迹分析。这些结果表明，p54.5-6.6是一种 ETS1蛋白的候选靶基因产物。进一步识别 P54.5-6.6蛋白正在研究中。 大量表达ETS1蛋白的克隆S7致瘤性降低 在软琼脂集落形成实验和裸鼠实验中。至 确认肿瘤致瘤性降低是由于ETS1在 DLD-1细胞，其他克隆正在通过独立的转染法进行筛选。

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.