Specific down-regulation of HER-2/neu mediated by a chimeric U6 hammerhead ribozyme results in growth inhibition of human ovarian carcinoma.
The U6 expression system was explored for efficient expression of a ribozyme against the human proto-oncogene c-neu. A hammerhead ribozyme (neuRz) and the control mutant ribozyme (MRz) were targeted to cleave c-neu mRNA at the tyrosine kinase domain. In vitro cleavage showed that neuRz was very active while MRz was not. Near-maximal target cleavage observed at a low ribozyme:target ratio (0.1) suggests that neuRz has good activity and turnover capability under physiological conditions, i.e., <5 mM MgCl(2) and 37 degrees C. Chimeric U6 ribozyme was expressed at about 5 x 10(6) copies/cell at 48 h in the ovarian carcinoma cell line SKOV-3.ip1. Partial down-regulation of c-neu mRNA and protein was observed in a dose-dependent manner in cells transiently transfected with U6neuRz- but not with MRz-containing plasmid. Sorted transient transfectants demonstrated dramatic growth inhibition with the neuRz-expressing cells. Our results demonstrate that the U6 expression system is very efficient and suitable for the expression of a hammerhead ribozyme. Moreover, nonviral delivery of the neuRz-expressing plasmid resulted in specific down-regulation of c-neu and, subsequently, growth inhibition of ovarian cancer cells overexpressing c-neu.
Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA.
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D015152:Blotting, Northern; D002455:Cell Division; D002469:Cell Separation; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005260:Female; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D010051:Ovarian Neoplasms / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D011505:Protein-Tyrosine Kinases; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; C000715470:MAS1 protein, human