Molecular and Cellular Characterization of Baboon C-Raf as a Target for Antiproliferative Effects of Antisense Oligonucleotides

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Abstract
C-Raf is a an essential member of the growth factor-ras pathway and a target for intervention strategies aimed at blocking cell proliferative responses. Excessive smooth muscle proliferation is considered one cause of the arterial closure in restenosis. Because of the similarity to the human cardiovascular system, a useful current animal model of the disease is a baboon model. As a foundation for animal studies employing antisense oligonucleotides, efforts were made to characterize the molecular and cellular biology of the baboon system. The nucleotide sequence of baboon c-raf cDNA was determined. Antisense phosphorothioate oligonucleotides specific to the 3′-UTR of c-raf mRNA from human and baboon were compared using primary baboon smooth muscle cells in culture. A particular human antisense oligonucleotide, referred to as ISIS 5132, was different by only 2 of 20 bases from the baboon sequence. The corresponding baboon antisense oligonucleotide ISIS 12959, however, was markedly more effective to inhibit c-raf mRNA, protein production, and DNA synthesis, and the results attest to the species specificity of the approach. After antisense treatment, c-raf mRNA levels dropped rapidly, whereas protein levels decreased with a half-life of roughly 24-48 hours, consistent with the antiproliferative effects. The data are discussed with regard to the profile of protein-protein interactions made by C-Raf and with the view that the baboon system closely parallels the human one at the signal transduction level. As this work progressed, a baboon cDNA homolog of a human c-raf-2 pseudogene was isolated, sequenced, and shown to be transcribed into mRNA.