Ribozyme-Mediated Induction of Apoptosis in Human Cancer Cells by Targeted Repair of Mutant p53 RNA

Abstract

A variety of mutations in the p53 tumor suppressor gene have been found in over half of human tumors. Thus, restoration of wild-type p53 activity by repair of mutant RNA has been previously suggested as an approach to cancer treatment. To explore the potential utility of RNA repair for cancer therapy, we developed a group I intron-based ribozyme that can replace mutant p53 RNA with a wild-type RNA sequence attached to the 3′ end of the ribozyme by trans-splicing reaction. First, RNA mapping analysis demonstrated that the leader sequences upstream of the AUG start codon in the mutant p53 RNA appeared to be particularly accessible to the ribozymes. Then, the trans-splicing ribozyme specifically recognizing the most accessible sequence induced functional p53 activity, resulting in an 8- and a 2.6-fold induction of transactivation of p53-responsive promoters in two mutant p53-related ovarian cancer cell lines, SKOV3 cells and 2774 cells, respectively, by repairing defective p53 RNA. The repair efficiency of the mutant p53 RNA was almost 10% in 2774 cells. Moreover, the ribozyme activated the expression level of endogenous p21 and Bax genes in the cells. Furthermore, apoptosis was efficiently triggered in the human cancer cells transfected with the specific ribozyme. These results suggest that a trans-splicing ribozyme could be a potent anti-cancer agent that can revert the defective p53-related neoplastic phenotype.