IDENTIFICATION OF TARGET GENES FOR ETS-1 GENE PRODUCT

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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.