Document Detail


Hammerhead ribozyme-mediated cleavage of the fusion transcript NPM-ALK associated with anaplastic large-cell lymphoma.
MedLine Citation:
PMID:  12644020     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
OBJECTIVE: Approximately 60% of all anaplastic large-cell lymphomas (ALCL) contain a specific t(2;5)(p23;q35) chromosomal translocation leading to overexpression of NPM-ALK. As the chimeric tyrosine kinase is involved in tumorigenesis and pathogenesis of ALCL, we were interested to inhibit NPM-ALK expression using an exogenous and an endogenous ribozyme approach. METHODS: We designed five anti-ALK hammerhead ribozymes that were targeted to cleave the ALK proportion of NPM-ALK. The ribozyme with the highest cleavage activity was used as a modified RNA/DNA chimera (RZ1*) for transient transfection and as a self-splicing ribozyme vector (pRZ1) for endogenous expression. Ribozyme performance was tested in 293 cells (cotransfected with NPM-ALK) and in the ALCL cell line Karpas 299 by transient and stable transfection and Western blotting. The half-life time of NPM-ALK was determined by pulse-chase experiments. RESULTS: In vitro cleavage assays demonstrated different catalytic efficiencies depending on the targeted site of the substrate. Constant transfection of Karpas 299 cells with RZ1* for 96 hours did not lead to a significant reduction of NPM-ALK protein, presumably due to the long half-life of NPM-ALK (48 hours). In contrast, NPM-ALK protein expression was almost completely suppressed in transiently transfected 293 cells. Stable transfection of Karpas 299 cells with pRZ1 also resulted in significant reduction of NPM-ALK expression. CONCLUSION: These results suggest that ribozymes targeted against NPM-ALK are able to inhibit expression of this oncogenic kinase efficiently and will be a useful tool to analyze its role in the pathophysiology of ALCL.
Authors:
Gabriele Hübinger; Engelbert Wehnes; Liquan Xue; Stephan W Morris; Ulrich Maurer
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Experimental hematology     Volume:  31     ISSN:  0301-472X     ISO Abbreviation:  Exp. Hematol.     Publication Date:  2003 Mar 
Date Detail:
Created Date:  2003-03-19     Completed Date:  2003-05-09     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  0402313     Medline TA:  Exp Hematol     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  226-33     Citation Subset:  IM    
Copyright Information:
Copyright 2003 International Society for Experimental Hematology
Affiliation:
Department of Internal Medicine III, University of Ulm, Ulm, Germany. gabi.huebinger@gmx.de
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Base Sequence
Cell-Free System
Humans
Hydrolysis
Lymphoma, Large-Cell, Anaplastic / genetics*
Molecular Sequence Data
Oncogene Proteins, Fusion / genetics
Protein-Tyrosine Kinases / genetics*
RNA, Catalytic / chemical synthesis,  genetics,  metabolism*
RNA, Messenger / metabolism*
Transfection
Tumor Cells, Cultured
Grant Support
ID/Acronym/Agency:
CA21765/CA/NCI NIH HHS; CA69129/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/Oncogene Proteins, Fusion; 0/RNA, Catalytic; 0/RNA, Messenger; 0/hammerhead ribozyme; EC 2.7.1.-/p80(NPM-ALK) protein; EC 2.7.10.1/Protein-Tyrosine Kinases

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


Previous Document:  Cycling B-CLL cells are highly susceptible to inhibition of the proteasome: involvement of p27, earl...
Next Document:  Mechanisms of anemia in SHP-1 protein tyrosine phosphatase-deficient "viable motheaten" mice.