Ribozyme-mediated suppression of platelet type 12 lipoxygenase in human erythroleukemia cells.
The platelet type 12 lipoxygenase (12-LOX) adds molecular oxygen to C-12 arachidonic acid to yield 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. It has been suggested that 12-LOX and its metabolites play an important role in tumor angiogenesis. A hammerhead ribozyme (Rz) targeted to the first GUC site within the 12-LOX mRNA was designed and cloned into an in vitro transcriptional or mammalian expression vector. In vitro, the Rz was able to cleave its substrate efficiently in a time-dependent manner. Under multiple turnover conditions, the Rz performed well at 37 degrees C, with a further improvement at 50 degrees C. When cloned into a mammalian expression vector, pSV2neo, the Rz construct efficiently decreased the level of 12-LOX mRNA in stably transfected human erythroleukemia cells to levels that were undetectable by Northern blot analyses. 12-LOX enzyme activity assays showed that Rz significantly reduced the 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid production in human erythroleukemia cells; this effect was sustained for up to 6 months in cell culture. The Rz developed in this study may represent a powerful tool for potential applications, ranging from an understanding of tumor angiogenesis to cancer gene therapy.
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Department of Molecular Virology and Bioengineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China.
Sun
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Tien
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D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism*; D001792:Blood Platelets / Q000201:enzymology*; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D006801:Humans; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology* / Q000378:metabolism; D008957:Models, Genetic; D009389:Neovascularization, Pathologic; D016337:RNA, Catalytic / Q000378:metabolism / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D016337:RNA, Catalytic; D012333:RNA, Messenger; D001092:Arachidonate 12-Lipoxygenase