title: Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture.
abstract: 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.
authors.affiliation: Medizinische Klinik II, Universitätsklinikum, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany.
authors.forename: C D
authors.initials: CD
authors.lastname: Giannini
authors.forename: W K
authors.initials: WK
authors.lastname: Roth
authors.forename: A
authors.initials: A
authors.lastname: Piiper
authors.forename: S
authors.initials: S
authors.lastname: Zeuzem
mesh_terms: D004789:Enzyme Activation / Q000235:genetics; D005786:Gene Expression Regulation; D011905:Genes, ras; D006801:Humans; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D016376:Oligonucleotides, Antisense; D016337:RNA, Catalytic