THE MITOGENIC RESPONSE OF T-CELLS TO INTERLEUKIN-2 REQUIRES RAF-1

The product of the c-raf-1 proto-oncogene, Raf-1, is known to encode a 74-kDa ubiquitously expressed cytoplasmic serine/threonine kinase. Va rious growth factors such as epidermal growth factor., acidic fibrobla st growth factor, platelet-derived growth factor, insulin, granulocyte -macrophage colony-stimulating factor, interleukin (IL)-2, IL-3 and er ythropoietin have been shown to induce phosphorylation of Raf-1, there by activating Raf-1 kinase. Raf-1 is, thus, believed to play a role in coupling growth factor receptors to proliferation. We have examined t he role of Raf-1 in the mitogenic response of human peripheral blood-d erived IL-2 receptor expressing T cells to human recombinant IL-2 empl oying c-raf antisense (AS) oligodeoxyribonucleotide. Uptake studies of oligonucleotides indicated that incorporation of oligomers was maxima l at 4 h and oligdeoxynucleotides remained stable in these cells for u p to 24 h. Treatment of T cells with the AS oligodeoxyribonucleotide i n intracellular duplex formation followed by efficient translation blo ckade of c-raf-1. In contrast, sense (S) and nonsense (NS) oligodeoxyn ucleotides failed to form intracellular duplexes and did not interfere with translation of c-raf-1, suggesting specific elimination of c-raf -1 by the AS oligomer. Proliferation of T cells ([H-3]thymidine incorp oration) following exposure to IL-2 was substantially reduced when the c-raf-1 AS oligodeoxyribonucleotide was added to cultures, while the mitogenic response to this factor remained almost unaffected in the pr esence of S and NS oligodeoxyribonucleotides.