Cioffi C L, Garay M, Johnston J F, McGraw K, Boggs R T, Hreniuk D, Monia B P
Research Department, CIBA-GEIGY Corporation, Summit, New Jersey 07901, USA.
Mol Pharmacol. 1997 Mar;51(3):383-9.
Raf kinases, cytoplasmic serine/threonine protein kinases, have been proposed as important participants in mitogen-induced signal transduction. However, the precise role that Raf kinase isozymes play in cellular responses such as proliferation has not been resolved. The present study investigates the ability of antisense phosphorothioate oligodeoxynucleotides (ODNs), targeted against rat C-Raf and A-Raf kinases, to reduce gene expression and proliferation of cultured rat A10 smooth muscle 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 completely dependent on ODN sequence because the incorporation of increasing numbers of mismatches (up to six) into the sequence resulted in sequential loss of potency. Similarly, a dose-dependent reduction (IC50 = 125 nM) in A-Raf gene expression was observed after treatment of cells with the active A-Raf ODN, ISIS 9069, whereas two scrambled controls were without effect. These results demonstrate that ISIS 11061 and ISIS 9069 reduced gene expression in a sequence-specific and isozyme-specific manner. Moreover, administration of ISIS 11061 and ISIS 9069 to rat SMCs resulted in a significant and potent diminution of serum-induced proliferation with corresponding IC50 values of 216 and 273 nM, respectively. Taken together, these results indicate that A-Raf and C-Raf kinases play an important role in regulating vascular SMC proliferation and that antisense-mediated inhibition of Raf kinase activity may serve as a therapeutic modality in the treatment of vascular proliferative disorders.
Raf激酶是一种胞质丝氨酸/苏氨酸蛋白激酶，被认为是有丝分裂原诱导信号转导的重要参与者。然而，Raf激酶同工酶在细胞增殖等细胞反应中的确切作用尚未明确。本研究探讨了针对大鼠C-Raf和A-Raf激酶的反义硫代磷酸酯寡脱氧核苷酸（ODN）降低培养的大鼠A10平滑肌细胞（SMC）基因表达和增殖的能力。将A10细胞暴露于活性C-Raf反义ODN ISIS 11061，可导致C-Raf mRNA和蛋白表达受到强效、剂量依赖性抑制（IC50 = 55 nM）。这种抑制完全依赖于ODN序列，因为在序列中引入越来越多的错配（最多六个）会导致活性逐渐丧失。同样，用活性A-Raf ODN ISIS 9069处理细胞后，观察到A-Raf基因表达呈剂量依赖性降低（IC50 = 125 nM），而两个随机对照则无此作用。这些结果表明，ISIS 11061和ISIS 9069以序列特异性和同工酶特异性方式降低基因表达。此外，将ISIS 11061和ISIS 9069施用于大鼠SMC可导致血清诱导的增殖显著且强效降低，相应的IC50值分别为216和273 nM。综上所述，这些结果表明A-Raf和C-Raf激酶在调节血管SMC增殖中起重要作用，并且反义介导的Raf激酶活性抑制可能作为治疗血管增殖性疾病的一种治疗方式。
