Ribozymes Targeting Telomerase

Telomerase is an exploitable target for strategies based on the use of antisense oligonucleotides. In fact, the template region of hTR, which naturally binds to the 3' single-strand overhang of the telomere end to add new telomeric repeats, is inherently accessible to incoming nucleic acids and represents a suitable target site for these approaches. A number of studies on experimental human tumor models have shown the possibility to obtain efficient inhibition of telomerase through the use of hammerhead ribozymes targeting hTR. The first developed hammerhead ribozyme was engineered to target a consensus sequence located at the end of the telomerase template (65). When added to cell extracts from two hepatocellular carcinoma cell lines (HepG2 and Huh-7), the ribozyme induced dose-dependent inhibition of telomerase activity. The potential use of chemically stabilized hammerhead ribozymes to inhibit telomerase activity by cleaving the hTR component was also pursued. Specifically, hammerhead ribozymes containing 2'-0-methyl ribonucleotides for enhanced biologic stability and designed to be complementary to the RNA component of human telomerase exhibited dose-dependent inhibition of telomerase activity in human glioma cell lysates and induced the cleavage of the full-length hTR in intact U87-MG cells (66).

The catalytic sequence described by Kanazawa et al. (64) was exploited in our laboratory to downregulate telomerase activity in intact human tumor cells. Specifically, the JR8 human melanoma cells were transfected with the ribozyme sequence inserted into a mammalian expression vector (67). Ribozyme transfec-tants successfully expressing the ribozyme and characterized by reduced telom-erase activity and a decreased level of telomerase RNA expression compared with mock transfectants were selected. Ribozyme-expressing clones grew more slowly than parental cells and also expressed an altered morphology with a dendritic appearance in monolayer cultures. A small but significant fraction of the cell population also expressed an apoptotic phenotype. However, no telom-ere shortening was observed in these clones even after a prolonged period (50 d) of growth in culture (67).

In a further study, three hammerhead ribozymes targeting GUC sequences from the 5'-end of telomerase RNA were described (68). In a cell-free system, all the ribozymes efficiently cleaved the RNA substrate. However, when the ribozymes were introduced into intact endometrial carcinoma Ishikawa cells, only the ribozyme targeting the template region was able to attenuate telom-erase activity. The ribozyme sequence was then inserted into an expression vector subsequently used to transfect the endometrial carcinoma cell line AN3CA. Ribozyme-expressing clones obtained after in vitro selection showed reduced telomerase activity and telomerase RNA expression. A marked reduction of telomere length was observed in some of these clones. However, even after 30 passages in vitro, these cells still maintained their ability to proliferate. To search for more potent ribozymes targeting telomerase, the same group recently reported the use of a divalent ribozyme (referred to as 36- to 59-divalent ribozyme) designed to cleave simultaneously the GUC triplet (which represents the most exploited target site in the template region of hTR) and the closest target sequence GUA, located 23 nt downstream from the GUC in the template region of hTR (69). Data obtained by in vitro cleavage assay showed that the 36- to 59-divalent ribozyme cleaved telomerase RNA more efficiently than the related monovalent ribozymes (36- and 59-ribozyme). However, when the divalent ribozyme was introduced into Ishikawa endometrial carcinoma cells, its inhibitory effect on telomerase activity was less than that of the 36-ribozyme, whereas the 59-ribozyme did not show a significant activity on telomerase.

Recently, hammerhead ribozymes were designed against seven NHH sequences located in open loops of the hTR secondary structure and introduced through an expression vector into human breast tumor MCF-7 cells. Results showed that stable transfectants of ribozyme R1 targeting the template region of hTR induced the degradation of target and attenuated telomerase activity in breast cancer 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 and the occurrence of apoptotic cells was observed (70).

Because the expression of hTERT is almost completely confined to tumor cells and its presence represents the rate-limiting step for telomerase activity, the antisense-mediated attenuation of hTERT mRNA expression would represent an excellent means to regulate the enzyme's activity in cancer cells (14). However, hTERT is a more challenging target than hTR for antisense-based strategies. In fact, its mRNA possesses a complex secondary structure that makes it difficult to accurately predict which target site will be most accessible for hybridization. As a consequence, there are still few studies based on the use of antisense-mediated approaches to achieve telomerase inhibition through hTERT downregulation.

However, the possibility to downregulate telomerase activity by the use of hammerhead ribozymes that target the mRNA of hTERT has been exploited. Specifically, seven presynthesized ribozymes, directed against 5'-NHH-3' consensus sequences within the hTERT mRNA, were delivered into endometrial carcinoma cells by cationic lipids and demonstrated to significantly inhibit telomerase activity in intact cells (71). However, a stable transfection of endometrial carcinoma cells carried out by cloning the ribozyme sequences into expression vectors confirmed the ability of only one ribozyme to suppress telomerase activity. In another study, Ludwig et al. (72) developed a hammerhead ribozyme directed against the consensus sequence within the T-motif of the hTERT mRNA that was able to attenuate telomerase activity in stable trans-fected clones of the immortal, telomerase-positive human breast epithelial cell line HBL-100 and the breast cancer cell line MCF-7. After a significant lag phase, in ribozyme-expressing clones the decline of the enzyme's catalytic activity resulted in telomere shortening, inhibition of cell proliferation, and induction of apoptosis. In addition, such clones demonstrated an increased susceptibility to topoisomerase II inhibitors such as doxorubicin, etoposide, and mitoxantrone. Successively, the same ribozyme sequence was transduced through an adenoviral vector into four ovarian cancer cell lines with widely different telomere lengths (73). The authors observed massive cell loss in mass cultures from all cell lines tested 3 d after transduction.

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