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.