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Specific down-regulation of HER-2/neu mediated by a chimeric U6 hammerhead ribozyme results in growth inhibition of human ovarian carcinoma.

Abstract
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.
AuthorsV W Lui, Y He, L Huang
JournalMolecular therapy : the journal of the American Society of Gene Therapy (Mol Ther) Vol. 3 Issue 2 Pg. 169-77 (Feb 2001) ISSN: 1525-0016 [Print] United States
PMID11237673 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
Chemical References
  • MAS1 protein, human
  • Proto-Oncogene Mas
  • RNA, Messenger
  • Ribonucleoprotein, U4-U6 Small Nuclear
  • Protein-Tyrosine Kinases
  • Receptor, ErbB-2
Topics
  • Blotting, Northern
  • Cell Division
  • Cell Separation
  • Dose-Response Relationship, Drug
  • Down-Regulation
  • Female
  • Flow Cytometry
  • Genetic Therapy (methods)
  • Humans
  • Ovarian Neoplasms (metabolism)
  • Plasmids (metabolism)
  • Promoter Regions, Genetic
  • Protein Structure, Tertiary
  • Protein-Tyrosine Kinases (chemistry)
  • Proto-Oncogene Mas
  • RNA, Messenger (metabolism)
  • Receptor, ErbB-2 (metabolism)
  • Ribonucleoprotein, U4-U6 Small Nuclear (metabolism)
  • Time Factors
  • Transfection
  • Tumor Cells, Cultured

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