Adenoviral-mediated Delivery of anti RET Hammerhead Ribozymes Inhibits Thyroid Medullary Carcinoma Cell Proliferation.

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Activating mutations of the RET proto-oncogene cause multiple endocrine neoplasia (MEN) 2A, MEN2B, familial medullary thyroid carcinoma (MTC). Down-regulation of RET expression may be an important strategy for cancer therapy. Indeed, we previously reported ribozyme targeting mutant RET suppress transformation of NIH 3T3 cells transfected with mutant RET. However, the most of mutant sequences are not compatible with requirement for cleavage by hammerhead ribozyme. In this study, we first designed hammerhead ribozymes to cleave the normal RET transcript. We select two target sites for cleavage of ribozymes targeting intracellular and extracellular domain, respectively. In vitro cleavage assay demonstrates that these ribozymes can cleave the artificial target sequence specifically and efficiently. In order to achieve efficient gene delivery, we developed recombinant adenoviruses encoding these ribozymes driven by the cytomegalovirus promoter. Human MTC cell line TT was infected with the adenoviruses. Reduction of RET expression in RNA and protein level was observed in active ribozyme-expressing adenovirus-infected cells. Concordantly, cell proliferation was also suppressed in the active ribozyme expressing adenovirus-infected TT cells. In order to demonstrate specificity of the ribozymes, infection into TPC-1 cells which harbor ret/PTC-1 rearrangement (where intracellular domain of RET is fused with irrelevant protein) exhibit that only ribozyme targeting intracellular domain has significant but much less prominent effect on the cell proliferation. Finally, caspase-3 activity was elevated in active ribozyme-expressing adenovirus infected TT cells, indicating that apoptotic pathways are involved in the effects of the ribozymes. These results provide a new avenue for inhibition of RET by selective mRNA degradation with its potential therapeutic application.