Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture

Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA
Due to their mode of action, ribozymes show antisense effects in addition to their specific cleavage activity. In the present study we investigated whether a hammerhead ribozyme is capable of cleaving mutated Ki-ras mRNA in a pancreatic carcinoma cell line and whether antisense effects contribute to the activity of the ribozyme. A 2[prime]-O-allyl modified hammerhead ribozyme was designed to cleave specifically the mutated form of the Ki-ras mRNA (GUU motif in codon 12). The activity was monitored by RT-PCR on Ki-ras RNA expression by determination of the relative amount of wild type to mutant Ki-ras mRNA, by 5-bromo-2[prime]-deoxy-uridine incorporation on cell proliferation and by colony formation in soft agar on malignancy in the human pancreatic adenocarcinoma cell line CFPAC-1, which is heterozygous for the Ki-ras mutation. A catalytically inactive ribozyme was used as control to differentiate between antisense and cleavage activity and a ribozyme with random guide sequences as negative control. The catalytically active anti-Ki-ras ribozyme was at least 2-fold more potent in decreasing cellular Ki-ras mRNA levels, inhibiting cell proliferation and colony formation in soft agar than the catalytically inactive ribozyme. The catalytically active anti-Ki-ras ribozyme, but not the catalytically inactive or random ribozyme, increased the ratio of wild type to mutated Ki-ras mRNA in CFPAC-1 cells. In conclusion, both cleavage activity and antisense effects contribute to the activity of the catalytically active anti-Ki-ras hammerhead ribozyme. Specific ribozymes might be useful in the treatment of pancreatic carcinomas containing an oncogenic GTT mutation in codon 12 of the Ki-ras gene.
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Metadaten
Author:Claudio Detlef Giannini, W. Kurt Roth, Albrecht Piiper, Stefan Zeuzem
URN:urn:nbn:de:hebis:30-32944
URL:http://nar.oxfordjournals.org/cgi/content/abstract/27/13/2737
ISBN:1362-4962
Parent Title (English):Nucleic acids research
Publisher:Oxford Univ. Press
Place of publication:Oxford
Document Type:Article
Language:English
Date of Publication (online):2006/11/10
Year of first Publication:1999
Publishing Institution:Univ.-Bibliothek Frankfurt am Main
Release Date:2006/11/10
Volume:27
Issue:13
Pagenumber:8
First Page:2737
Last Page:2744
Note:
© 1999 by Oxford University Press, 
http://nar.oxfordjournals.org/misc/terms.dtl Online ISSN 1362-4962 - Print ISSN 0305-1048
HeBIS PPN:288128966
Institutes:Medizin
Georg-Speyer-Haus
Dewey Decimal Classification:610 Medizin und Gesundheit
Sammlungen:Universitätspublikationen
Licence (German):License Logo Veröffentlichungsvertrag für Publikationen

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