SELECTIVE-INHIBITION OF A-RAF AND C-RAF MESSENGER-RNA EXPRESSION BY ANTISENSE OLIGODEOXYNUCLEOTIDES IN RAT VASCULAR SMOOTH-MUSCLE CELLS - ROLE OF A-RAF AND C-RAF IN SERUM-INDUCED PROLIFERATION

Raf kinases, cytoplasmic serine/threonine protein kinases, have been p roposed as important participants in mitogen-induced signal transducti on. However, the precise role that Raf kinase isozymes play in cellula r responses such as proliferation has not been resolved. The present s tudy investigates the ability of antisense phosphorothioate oligodeoxy nucleotides (ODNs), targeted against rat C-Raf and A-Raf kinases, to r educe gene expression and proliferation of cultured rat A10 smooth mus cle cells (SMCs). Exposure of A10 cells to ISIS 11061, an active C-Raf antisense ODN, resulted in a potent, dose-dependent inhibition (IC50 = 55 nM) of C-Raf mRNA and protein expression. This inhibition was com pletely dependent on ODN sequence because the incorporation of increas ing numbers of mismatches (up to six) into the sequence resulted in se quential loss of potency. Similarly, a dose-dependent reduction (IC50 = 125 nM) in A-Raf gene expression was observed after treatment of cel ls with the active A-Raf ODN, ISIS 9069, whereas two scrambled control s were without effect. These results demonstrate that ISIS 11061 and I SIS 9069 reduced gene expression in a sequence-specific and isozyme-sp ecific manner. Moreover, administration of ISIS 11061 and ISIS 9069 to rat SMCs resulted in a significant and potent diminution of serum-ind uced proliferation with corresponding IC50 values of 216 and 273 nM, r espectively. Taken together, these results indicate that A-Raf and C-R af kinases play an important role in regulating vascular SMC prolifera tion and that antisense-mediated inhibition of Raf kinase activity may serve as a therapeutic modality in the treatment of vascular prolifer ative disorders.