Ribozymes Targeting Survivin

We first demonstrated the possibility to efficiently inhibit survivin expression through the use of ribozymes. Specifically, we designed two hammerhead ribozymes targeting the 3'-end of the CUA110 (RZ7) and the GUC294 (RZ1) triplets in the survivin mRNA and transfected them into the JR8 human melanoma cell line over expressing survivin. Stably transfected clones proven to endogenously express the active ribozyme RZ1 or RZ7 were characterized by a markedly lower survivin protein level than JR8 parental cells, whereas a negligible reduction of survivin expression was observed in cells expressing a mutant ribozyme (which was produced by introducing a mutation in the catalytic core of the active ribozyme RZ1). These cells demonstrated an increased caspase-9-dependent apoptotic response to cisplatin treatment (74). JR8 cells expressing RZ1 also showed a significantly increased sensitivity to the topoisomerase-I inhibitor topotecan (as detected by clonogenic cell survival) as a consequence of an enhanced rate of drug-induced caspase-9-dependent apoptosis. Moreover, an increased antitumor activity of oral topotecan was observed in ribozyme-express-ing JR8 cells grown as xenograft tumors in athymic nude mice (75). JR8 cells endogenously expressing the active RZ7 ribozyme also showed significantly increased sensitivity to y-irradiation (76). More recently, we constructed a Moloney-based retroviral vector expressing the RZ7 ribozyme, encoded as a chimeric RNA within adenoviral VA1 RNA. Polyclonal cell populations, obtained by infection with the retroviral vector, of two androgen-independent human prostate cancer cell lines (DU145 and PC-3) were characterized by a significant reduction of survivin expression; the cells became polyploid, underwent caspase-9-dependent apoptosis, and showed an altered pattern of gene expression, as detected by oligonucleotide array analysis. Survivin inhibition also increased the susceptibility of these cells to cisplatin-induced apoptosis and prevented tumor formation when cells were xenografted into athymic nude mice (77).

Choi et al. (78) recently showed that two hammerhead ribozymes, able to cleave the human survivin mRNA at nucleotide position +279 and +28 and cloned into a replication-deficient adenoviral vector, were able to increase the apoptotic response to etoposide in transduced MCF-7 breast cancer cells.

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