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 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.
Author: | Claudio Detlef Giannini, W. Kurt Roth, Albrecht PiiperORCiD, Stefan ZeuzemORCiDGND |
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URN: | urn:nbn:de:hebis:30-32944 |
DOI: | https://doi.org/10.1093/nar/27.13.2737 |
ISSN: | 1362-4962 |
ISSN: | 0305-1048 |
Pubmed Id: | https://pubmed.ncbi.nlm.nih.gov/10373591 |
Parent Title (English): | Nucleic acids research |
Publisher: | Oxford Univ. Press |
Place of publication: | Oxford |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 1999/07/01 |
Date of first Publication: | 1999/07/01 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2006/11/10 |
Volume: | 27 |
Issue: | 13 |
Page Number: | 8 |
First Page: | 2737 |
Last Page: | 2744 |
Note: | © 1999 Oxford University Press |
HeBIS-PPN: | 288128966 |
Institutes: | Medizin / Medizin |
Angeschlossene und kooperierende Institutionen / Georg-Speyer-Haus | |
Dewey Decimal Classification: | 6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit |
Licence (German): | Deutsches Urheberrecht |