Hammerhead ribozyme-mediated cleavage of the fusion transcript NPM-ALK associated with anaplastic large-cell lymphoma.
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.


CONCLUSIONS
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.
Hübinger
Gabriele
G
Department of Internal Medicine III, University of Ulm, Ulm, Germany. gabi.huebinger@gmx.de
Wehnes
Engelbert
E
Xue
Liquan
L
Morris
Stephan W
SW
Maurer
Ulrich
U
D001483:Base Sequence; D002474:Cell-Free System; D006801:Humans; D006868:Hydrolysis; D017728:Lymphoma, Large-Cell, Anaplastic / Q000235:genetics*; D008969:Molecular Sequence Data; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D016337:RNA, Catalytic / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D015514:Oncogene Proteins, Fusion; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases