Exposure of human vascular smooth muscle cells to Raf-1 antisense oligodeoxynucleotides: cellular responses and pharmacodynamic implications.
To characterize the pharmacodynamic properties of CGP 69846A/ISIS 5132, an antisense oligodeoxynucleotide directed against the mitogenic signal transducer Raf-1 kinase, we investigated the elicited biological responses in human coronary artery smooth muscle cells. Cell exposure to CGP 69846A resulted in a reversible time- and concentration-dependent down-regulation of cellular Raf-1 gene expression and, ultimately, inhibition of cell cycle progression. The highest potencies of this compound to reduce Raf-1 mRNA and protein levels were observed after 24 and 48 hr of cell exposure, respectively, with corresponding IC50 values of approximately 100 and approximately 300 nM. Proliferation was inhibited with an IC50 value of approximately 300 nM after 72 hr. We interpreted the recovery rate of Raf-1 mRNA after cell exposure to antisense ODNs as the half-life (t1/2 approximately 50 hr) of active intracellular CGP 69846A in our cell culture system. The endogenous Raf-1 turnover half-life of approximately 30 hr, as assessed by monitoring metabolically labeled Raf-1 protein, correlated kinetically with the antisense-induced protein decay rate (50% decay in approximately 33 hr), indicating that the efficiency of CGP 69846A in decreasing Raf-1 protein levels was rate-limited by the endogenous protein turnover rate. The pharmacodynamic effects of CGP 69846A antisense ODNs are therefore limited by the duration of its intracellular activity rather than by its ability to transiently decrease mRNA levels. Local steady state exposure to CGP 69846A may represent a new approach to prevent the transition of quiescent vascular smooth muscle cells into the pathologically hyperproliferating cells seen after angioplasty.
Schumacher
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Research Department, CIBA-GEIGY Corporation, Summit, New Jersey 07901, USA. schumacher@pharma.novartis.com
Cioffi
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Sharif
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Haston
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Monia
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Wennogle
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D000970:Antineoplastic Agents / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D019908:Proto-Oncogene Proteins c-raf / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects
D000970:Antineoplastic Agents; D020319:Oligodeoxyribonucleotides, Antisense; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D013873:Thionucleotides; C099587:ISIS 5132; D019908:Proto-Oncogene Proteins c-raf