Inhibition of telomerase activity by a hammerhead ribozyme targeting the RNA component of telomerase in human melanoma cells

Reactivation of telomerase, an RNA-dependent DNA polymerase that synthesize s new telomeric repeats at the end of chromosomes, is a very common feature in human cancers. Telomerase is thought to be essential in maintaining the proliferative capacity of tumor cells and, as a consequence, it could repr esent an attractive target for new anti-cancer therapies. In this study, we generated a hammerhead ribozyme composed of a catalytic domain with flanki ng sequences complementary to the RNA component of human telomerase and des igned to cleave specifically a site located at the end of the telomerase te mplate sequence. In vitro the ribozyme induced cleavage of a synthetic RNA substrate obtained by cloning a portion of the RNA component of human telom erase, The extent of cleavage was dependent on the ribozyme/substrate ratio as well as the Mg2+ concentration. Moreover, when added to cell extracts f rom two human melanoma cell lines (JR8 and M14), or three melanoma surgical specimens, the ribozyme inhibited telomerase activity in a concentration- and time-dependent manner. When the ribozyme was delivered to growing JR8 m elanoma cells by (N-(1-(2,3 dioleoxyloxy)propil)-N,N,N trimethylammonium me thylsulfate-mediated transfer, a marked inhibition of telomerase activity w as observed. Next, the ribozyme sequence was cloned in an expression vector and JR8 cells were transfected with it. The cell clones obtained showed a reduced telomerase activity and telomerase RNA levels and expressed the rib ozyme, Moreover, ribozyme transfectants had significantly longer doubling t imes than control cells and showed a dendritic appearance in monolayer cult ure. No telomere shortening, however, was observed in these clones. Overall , our results indicate that the hammerhead ribozyme is a potentially useful tool for the inactivation of telomerase in human tumors.