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 synthesizes 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 represent an attractive target for new anti-cancer therapies. In this study, we generated a hammerhead ribozyme composed of a catalytic domain with flanking sequences complementary to the RNA component of human telomerase and designed to cleave specifically a site located at the end of the telomerase template sequence. In vitro the ribozyme induced cleavage of a synthetic RNA substrate obtained by cloning a portion of the RNA component of human telomerase. The extent of cleavage was dependent on the ribozyme/substrate ratio as well as the Mg2+ concentration. Moreover, when added to cell extracts from 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 melanoma cells by (N-(1-(2,3 dioleoxyloxy)propil)-N,N,N trimethylammonium methylsulfate-mediated transfer, a marked inhibition of telomerase activity was 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 ribozyme. Moreover, ribozyme transfectants had significantly longer doubling times than control cells and showed a dendritic appearance in monolayer culture. 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.
Folini
M
M
Department of Experimental Oncology, National Cancer Institute, Milan, Italy.
Colella
G
G
Villa
R
R
Lualdi
S
S
Daidone
M G
MG
Zaffaroni
N
N
D006801:Humans; D010641:Phenotype; D012313:RNA; D016337:RNA, Catalytic / Q000494:pharmacology*; D019098:Telomerase / Q000037:antagonists & inhibitors* / Q000235:genetics*; D016615:Telomere / Q000235:genetics / Q000502:physiology; D014407:Tumor Cells, Cultured
D016337:RNA, Catalytic; C121505:Rz967; D012313:RNA; D019098:Telomerase