Nucleic Acids Research, Vol 27, Issue 13 2737-2744, Copyright © 1999 by Oxford University Press
CD Giannini, WK Roth, A Piiper and S Zeuzem
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.
ARTICLES
Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture
Medizinische Klinik II, Universitatsklinikum, Theodor-Stern-Kai 7, D- 60590 Frankfurt am Main, Germany.
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