The ETS1 proto-oncogene is expressed at a higher level in lymphoid T- cells. The expression of ETS1 is related to the expression of the alpha T-cell receptor (TCRalpha) during development. The expression of ETS1 is maintained in mature resting T cells, and its level decreases after activation. ETS1 is a transcription factor and binds to a purine-rich motif (AGGAAGT) found in the regulatory regions of many different genes, i.e., lymphokines, TCRalpha, FOS, ETS1, ETS2. To understand the function of ETS1 gene products in T-cells, the ETS1 activity is blocked by express- ing antisense ETS1 RNA. Antisense RNAs are able to form hybrids with sense RNA, thus blocking the formation of proteins. These isogenic transfectant cell lines will then be used to investigate the changes that occur downstream of ETS1 functional pathways. For this purpose, Jurkat T- cells were subcloned by limited dilution and used for transfection. The full-length ETS1 cDNA and subfragments (representing the 5' region of the cDNA) were cloned in cytomegalovirus-based expression vectors and episomal vectors to express antisense RNA. Jurkat cells were transfected with these expression vectors by electroporation. Independent clones were selected in G418 and are under further investigation. In particular, FACS and Northern analysis reflecting the expression pattern of surface antigen (TCRalpha, TCRbeta, CD3, CD4), the growth properties of the transfected cell (growth rate, tumorigenesis), the production of lymphokines by these cells (ELISA), and the modulation of specific candidate genes targeted by ETS1 (using Northern analysis and immunoprecipitation and CAT assays) are being performed in a series of comprehensive experiments.
