Attenuation of telomerase activity by hammerhead ribozyme targeting human telomerase RNA induces growth retardation and apoptosis in human breast tumor cells.
Ribozyme possesses specific endoribonuclease activity and catalyzes the hydrolysis of specific phosphodiester bonds, which results in the cleavage of target RNA sequences. Here, we evaluated the ability of hammerhead ribozymes targeting human telomerase RNA (hTR) to inhibit the catalytic activity of telomerase and the proliferation of cancer cells. Hammerhead ribozymes were designed against 7 NUX sequences located in open loops of the hTR secondary structure. We verified the ribozyme specificity by in vitro cleavage assay by using a synthetic RNA substrate. Subsequently, we introduced ribozyme expression vector into human breast tumor MCF-7 cells and assessed the biologic effects of ribozyme. Hammerhead ribozyme R1 targeting the template region of hTR efficiently cleaved hTR in vitro, and stable transfectants of this ribozyme induced the degradation of target hTR RNA and attenuated telomerase activity in MCF-7 cells. Moreover, the ribozyme R1 transfectant displayed a significant telomere shortening and a lower proliferation rate than parental cells. Clones with reduced proliferation capacity showed enlarged senescence-like shapes or highly differentiated dendritic morphologies of apoptosis. In conclusion, the inhibition of telomerase activity by hammerhead ribozyme targeting the template region of the hTR presents a promising strategy for inhibiting the growth of human breast cancer cells.
Yeo
Marie
M
Cancer Metastasis Research Center, Yonsei Cancer Center, Department of Internal Medicine, Yonsei University College of Medicine, Seoul, South Korea.
Rha
Sung Young
SY
Jeung
Hei Cheul
HC
Hu
Shen Xiong
SX
Yang
Sang Hwa
SH
Kim
Yang Seok
YS
An
Sung Whan
SW
Chung
Hyun Cheol
HC
D017209:Apoptosis; D001943:Breast Neoplasms / Q000473:pathology*; D049109:Cell Proliferation; D005260:Female; D006801:Humans; D012313:RNA / Q000096:biosynthesis / Q000378:metabolism*; D016337:RNA, Catalytic / Q000494:pharmacology*; D019098:Telomerase / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D012313:RNA; D019098:Telomerase