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Inhibition of Telomerase Activity by a Hammerhead Ribozyme Targeting the RNA Component of Telomerase in Human Melanoma Cells

2000; Elsevier BV; Volume: 114; Issue: 2 Linguagem: Inglês

10.1046/j.1523-1747.2000.00870.x

1523-1747

Marco Folini, Gennaro Colella, Raffaella Villa, Susanna Lualdi, Maria Grazia Daidone, Nadia Zaffaroni,

RNA Interference and Gene Delivery

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. 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. (N-(1-(2,3 dioleoxyloxy)propil)-N,N,N trimethylammonium methylsulfate telomeric repeat amplification protocol Human telomeres are specialized structures located at the end of chromosomes, consisting of simple repeated DNA sequences (TTAGGG)n and associated proteins. They are essential to protect chromosomes against exonucleolytic degradation and to prevent aberrant recombination that may cause DNA rearrangements leading to karyotypic changes and genomic instability (Blackburn, 1991Blackburn E.H. Structure and function of telomeres.Nature. 1991; 350: 569-573Crossref PubMed Scopus (2911) Google Scholar). It is known that, due to the ''end replication problem'' (Watson, 1972Watson J. Origin of concatemeric T7 DNA.Nat Biol. 1972; 239: 197-201Crossref Scopus (1298) Google Scholar;Olovnikov, 1973Olovnikov A.M. A theory of marginotomy: the incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon.J Theor Biol. 1973; 41: 181-190Crossref PubMed Scopus (1339) Google Scholar), telomeric DNA shortens during the proliferation of human somatic cells. The phenomenon is responsible for cell senescence that occurs by growth arrest as a response to the shortened telomeres (Harley, 1991Harley C.B. Telomere loss: mitotic clock or genetic time bomb?.Mutat Res. 1991; 256: 271-282Crossref PubMed Scopus (1063) Google Scholar;Allsopp et al., 1992Allsopp R.C. Vaziri H. Patterson C. et al.Telomere length predicts replicative capacity of human fibroblasts.Proc Natl Acad Sci USA. 1992; 89: 10114-10118Crossref PubMed Scopus (1885) Google Scholar). To compensate for the telomeric sequences loss, germline, immortalized, and tumor cells express the RNA-dependent DNA polymerase telomerase (Counter et al., 1992Counter C.M. Avilion A.A. LeFeuvre C.E. Stewart N.G. Greider C.W. Harley C.B. Bacchetti S. Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity.EMBO J. 1992; 11: 1921-1929Crossref PubMed Scopus (1881) Google Scholar;Kim et al., 1994Kim N.W. Piatyszek M.A. Prowse K.R. et al.Specific association of human telomerase activity with immortal cells and cancer.Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6308) Google Scholar;Chadeneau et al., 1995Chadeneau C. Hay K. Hirte H.W. Gallinger S. Bacchetti S. Telomerase activity associated with acquisition of malignancy in human colorectal cancer.Cancer Res. 1995; 55: 2533-2536PubMed Google Scholar;Greider, 1998Greider C.W. Telomerase activity, cell proliferation, and cancer.Proc Natl Acad Sci USA. 1998; 95: 90-92Crossref PubMed Scopus (353) Google Scholar). The enzyme is a ribonucleoprotein which contains a short RNA molecule that serves as a template for the synthesis of new telomeric sequences (Collins, 1996Collins K. Structure and function of telomerase.Curr Opin Cell Biol. 1996; 8: 374-380Crossref PubMed Scopus (37) Google Scholar). The enzyme is involved in the attainment of immortality in cancer cells and therefore may contribute to tumorigenesis and neoplastic progression (Autexier and Greider, 1996Autexier C. Greider C.W. Telomerase and cancer: revisiting the telomere hypothesis.Trends Biochem Sci. 1996; 21: 387-391https://doi.org/10.1016/0968-0004(96)10042-6Crossref PubMed Scopus (166) Google Scholar). Thus far, telomerase activity has been detected in about 90% of human tumors screened (Shay and Bacchetti, 1997Shay J.W. Bacchetti S. A survey of telomerase activity in human cancer.Eur J Cancer. 1997; 33: 787-791https://doi.org/10.1016/s0959-8049(97)00062-2Abstract Full Text PDF PubMed Scopus (0) Google Scholar). Owing to its pattern of peculiar expression, telomerase has been proposed as a new diagnostic tool (Sugino et al., 1996Sugino T. Yoshida K. Bolodeoku J. et al.Telomerase activity in human breast cancer and benign breast lesions: diagnostic applications in clinical specimens, including fine needle aspirates.Int J Cancer. 1996; 69: 301-306https://doi.org/10.1002/(sici)1097-0215(19960822)69:4 3.0.co;2-8Crossref PubMed Scopus (0) Google Scholar;Villa et al., 1998Villa R. Zaffaroni N. Folini M. Martelli G. De Palo G. Daidone M.G. Silvestrini R. Telomerase activity in benign and malignant breast lesions: a pilot prospective study on fine-needle aspirates.J Natl Cancer Inst. 1998; 90: 537-539Crossref PubMed Scopus (27) Google Scholar), as a target for new anti-cancer therapies (Raymond et al., 1996Raymond E. Sun D. Chen S.-F. Von Windle B. Hoff D.D. Agents that target telomerase and telomeres.Curr Opin Biotech. 1996; 7: 583-591Crossref PubMed Scopus (81) Google Scholar) and, possibly, as a putative prognostic factor (Clark et al., 1997Clark G.M. Osborne C.K. Levitt D. Wu F. Kim N.W. Telomerase activity and survival of patients with node-positive breast cancer.J Natl Cancer Inst. 1997; 89: 1874-1881Crossref PubMed Scopus (138) Google Scholar;Hoos et al., 1998Hoos A. Hepp H.H. Kaul S. Ahlert T. Bastert G. Wallwiener D. Telomerase activity correlates with tumor aggressiveness and reflects therapy effect in breast cancer.Int J Cancer. 1998; 79: 8-12https://doi.org/10.1002/(sici)1097-0215(19980220)79:1 3.0.co;2-5Crossref PubMed Scopus (0) Google Scholar). One possible strategy to identify anti-telomerase compounds is based on the screening of molecules that can interact with the ribonucleoprotein components. Specifically, taking advantage of structural and functional similarities in telomerase and retroviral reverse transcriptase (Lingner et al., 1997Lingner J. Hughes T.R. Shevchenko A. Mann M. Lundblad V. Cech T.R. Reverse transcriptase motifs in the catalytic subunit of telomerase.Science. 1997; 276: 561-567Crossref PubMed Scopus (1003) Google Scholar;Nakamura and Cech, 1998Nakamura T.M. Cech T.R. Reversing time: origin of telomerase.Cell. 1998; 92: 587-590Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar), reverse transcriptase inhibitors, such as nucleoside analogs, have been also used as inhibitors of telomerase activity (Strahl and Blackburn, 1996Strahl C. Blackburn E.H. Effects of reverse transcriptase inhibitors on telomere length and telomerase activity in two immortalized human cell lines.Mol Cell Biol. 1996; 16: 53-65Crossref PubMed Scopus (348) Google Scholar;Yegorov et al., 1996Yegorov Y.E. Chernov D.N. Akimov S.S. Bolsheva N.L. Krayevsky A.A. Zelenin A.V. Reverse transcriptase inhibitors suppress telomerase function and induce senescence-like processes in cultured mouse fibroblasts.FEBS Lett. 1996; 389: 115-118https://doi.org/10.1016/0014-5793(96)00533-9Abstract Full Text PDF PubMed Scopus (79) Google Scholar;Melana et al., 1998Melana S.M. Holland J.F. Pogo B.G.-T. Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by 3′-azido-3′-deoxythymidine.Clin Cancer Res. 1998; 4: 693-696PubMed Google Scholar). An additional targeted strategy deals with the physical blockage of the RNA template. In fact, it has been shown that chemically modified oligonucleotides, such as phosphorothioate oligomers and peptide nucleic acids, efficiently inhibit telomerase activity in vitro (Norton et al., 1996Norton J.C. Piatyszek M.A. Wright W.E. Shay J.W. Corey D.R. Inhibition of human telomerase activity by peptide nucleic acids.Nat Biotechnol. 1996; 14: 615-619Crossref PubMed Scopus (344) Google Scholar). A marked reduction of telomerase catalytic activity was also obtained by using hammerhead ribozymes directed against the RNA component of the enzyme (Kanazawa et al., 1996Kanazawa Y. Ohkawa K. Ueda K. et al.Hammerhead ribozyme-mediated inhibition of telomerase activity in extracts of human hepatocellular carcinoma cells.Biochem Biophys Res Commun. 1996; 225: 570-576https://doi.org/10.1006/bbrc.1996.1213Crossref PubMed Scopus (101) Google Scholar;Yokoyama et al., 1998Yokoyama Y. Takahashi Y. Shinohara A. Wan X. Niwwa K. Tamay A. Attenuation of telomerase activity by a hammerhead ribozyme targeting the template region of the telomerase RNA in endometrial carcinoma cells.Cancer Res. 1998; 59: 5406-5410Google Scholar). Ribozymes are RNA molecules which possess specific endoribonuclease activity and catalyze the hydrolysis of specific phosphodiester bonds, resulting in the cleavage of the RNA target sequences (Irie et al., 1997Irie A. Kijima H. Ohkawa T. et al.Anti-oncogene ribozymes for cancer gene therapy.Adv Pharmacol. 1997; 40: 207-257Crossref PubMed Scopus (18) Google Scholar;James and Gibson, 1998James H.A. Gibson I. The therapeutic potential of ribozymes.Blood. 1998; 91: 371-382Crossref PubMed Google Scholar). Moreover, after the cleavage reaction, the substrate is accessible by ribonucleases, a step that guarantees its permanent inactivation and offers a considerable advantage over the simple physical blockage obtained with complementary oligomers. In this study, we evaluated the ability of a hammerhead ribozyme, directed against the RNA component of human telomerase, to inhibit the catalytic activity of the enzyme. We first verified the ribozyme specificity, in terms of its ability to cleave in vitro a synthetic RNA substrate that corresponds to a portion of telomerase RNA component. Subsequently, the efficiency of the ribozyme in inhibiting telomerase activity was assessed on protein extracts from telomerase-positive cell lines and surgical specimens of human cutaneous melanoma. Lastly, we examined whether cationic liposome as well as expression vector-mediated transfer of the ribozyme inhibited telomerase activity and induced changes in the proliferative and phenotypic features of melanoma cells growing in culture. Two cell lines (JR8 and M14) and three surgical specimens (ML1528, ML1583, ML1584) of human cutaneous melanoma were used in the study. The biologic characteristics of the cell lines have been previously reported (Zupi et al., 1985Zupi G. Mauro F. Balduzzi M.A. Pardini C. Cavaliere R. Greco C. Established melanoma cell lines from different metastatic nodules of a single patient. A useful model for cancer therapy.Proc Am Assoc Cancer Res. 1985; 26: 22Google Scholar). JR8 and M14 cells were maintained in logarithmic growth phase at 37°C in a 5% CO2 humidified atmosphere using RPMI 1640 (Biowhittaker, Verviers, Belgium) supplemented with 10% fetal calf serum, 2 mM L-glutamine and 0.25% gentamycin. Single-stranded synthetic DNA oligonucleotides encoding active ribozyme (telo RZ –) and negative control (telo RZ +) were obtained from M-Medical S.r.l. (Firenze, Italy). The oligonucleotide sequences were the following: telo-RZ – (5′-AGCTTGGCGGCCGCCATTTTTTGTTTCGTCCTCAC-GACTCTTCAGTAACCCTAACGCGGCCGCG-3′) and telo-RZ + (5′- AATTCGCGGCCGCGTTAGGGTTACTGAAGAGTCCGTGA-GGACGAAACAAAAAATGGCGGCCGCCA-3′). Annealing of the complementary oligonucleotides produced a fragment with EcoRI and HindIII protruding ends. The fragment was inserted into the pGEM-3Z vector (Promega, Madison, WI), which had been previously digested with HindIII and EcoRI restriction enzymes. The presence and the correct orientation of the insert was verified by DNA sequencing (AmpliCycle, Perkin Elmer-Roche Molecular System, Branchburg, NJ). The resulting plasmid was named pRZ. To obtain the active ribozyme (RZH) and the negative control oligomer, we linearized the pRZ plasmid with HindIII and EcoRI, respectively. Twenty micrograms of the resulting linear templates were transcribed in vitro in 0.2 ml of a reaction mixture composed of 260 U recombinant RNAsin ribonuclease inhibitor (Promega), 1 mM each of ribonucleotide triphosphates, 10 mM dithiothreitol (DTT), 0.1 mg per ml bovine serum albumin, 1 × transcription optimized buffer (40 mM Tris–HCl, pH 7.9, 10 mM NaCl, 6 mM MgCl2, 2 mM spermidine) and 300 U T7 RNA polymerase for RZH and 300 U SP6 RNA polymerase for negative control oligomer (Figure 1). All reagents were purchased from Promega. The reaction mixtures were incubated for 2 h at 37°C (RZH) or 40°C (negative control oligomer), as suggested bySambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, a Laboratory Manual.Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, 1989Google Scholar, then treated with 20 U RQ1 RNase-free DNase for 15 min at 37°C. The products were extracted with phenol/chloroform saturated with 10 mM Tris-1 mM ethylenediaminetetraacetic acid (EDTA), pH 4.5, precipitated with 100% isopropyl alcohol/5 M ammonium acetate, pH 5.5, washed with 70% pre-chilled ethanol, dried by speed vacuum and resuspended in twice distilled water. The concentration and the purity of the transcription products were assessed by A260 and A260/A280 spectrophotometrical measurement, respectively. The transcription products of expected size were then verified by denaturating polyacrylamide gel electrophoresis. To produce a synthetic RNA substrate, total RNA was isolated from the JR8 melanoma cell line using a Qiagen total RNA kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. A portion of the human telomerase RNA component containing the telomeric template element (Feng et al., 1995Feng J. Funk W.D. Wang S.-S. et al.The RNA component of human telomerase.Science. 1995; 269: 1236-1241Crossref PubMed Scopus (2018) Google Scholar) was produced by reverse transcriptase–polymerase chain reaction (reverse transcriptase–PCR). Reverse transcription was done using a GeneAmp RNA PCR core kit (Perkin Elmer) according to the manufacturer's instructions. The resulting cDNA was amplified using telomerase RNA sequence specific primers (telo S, 5′-CTGGGAGGGGTGGTGGCCAT-3′ and telo A, 5′-GGAGCAAAAG-CACGGCGCCT-3′ (Kanazawa et al., 1996Kanazawa Y. Ohkawa K. Ueda K. et al.Hammerhead ribozyme-mediated inhibition of telomerase activity in extracts of human hepatocellular carcinoma cells.Biochem Biophys Res Commun. 1996; 225: 570-576https://doi.org/10.1006/bbrc.1996.1213Crossref PubMed Scopus (101) Google Scholar)) and by performing 26 cycles of PCR (95°C for 30 s, 65°C for 45 s, and 72°C for 30 s) followed by a 5 min extension step at 72°C. The product of expected size was gel purified and cloned into pGEM-T-Easy vector (Promega) according to the manufacturer's instructions. The presence and the orientation of the fragment were verified by DNA sequencing. The resulting plasmid was named pRNAtelo. To obtain the internally labelled RNA substrate, pRNAtelo was linearized with NcoI restriction enzyme. The resulting linear template was transcribed in vitro in the presence of [α-32P]cytidine triphosphate (10 μCi per μl, 800 Ci per mmol, Amersham International, Buckinghamshire, U.K.) and SP6 RNA polymerase, using the Riboprobe in vitro transcription system (Promega) according to the manufacturer's instructions. The reaction product was purified and the transcription product of expected size was verified as described above. 32P-substrate RNA (0.1 μM) was mixed with increasing concentrations of ribozyme in 50 mM Tris–HCl, pH 7.5. The mixtures were heated for 2 min at 90°C, cooled at 37°C, and then incubated in the presence of increasing concentrations of MgCl2. The cleavage reactions were performed at 37°C for 2 h and at 23°C for 12 h. Reactions were stopped by adding an equal volume of stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.02% xylene cyanol FF). Products were resolved on 10% polyacrylamide/8 M urea gel, and results were quantitated by densitometric analysis. DOTAP (N-(1-(2,3 dioleoyloxy)propil)-N,N,N-trimethylammonium methyl sulfate, Boehringer Mannheim, Mannheim, Germany) and the RZH ribozyme, or the negative control oligomer, were mixed at various concentrations in 20 mM HEPES buffer, in a final volume of 1 ml, followed by incubation at room temperature for 15 min. Then, 100 μg per ml of recombinant RNasin ribonuclease inhibitor was added to the complex solution. For the liposome-mediated ribozyme transfection, 8 × 105 cells in 2 ml culture medium were plated in each well of a six-well plate and allowed to attach for 24 h at 37°C in a 5% CO2 humidified atmosphere. Cells were then incubated with DOTAP alone or with the DOTAP-ribozyme complexes. The final concentration of DOTAP alone was 60 μg per 2 ml per well and that of liposomes–ribozyme complexes was 60 μg DOTAP and 10–45 μg ribozyme/2 ml per well. After 48 h of incubation with DOTAP or the DOTAP–ribozyme complexes, cells were trypsinized and counted in a Coulter Counter (Coulter Electronics, Luton, U.K.), and their viability was determined by the Trypan Blue dye exclusion test. Cell extracts were then obtained (see below) for the determination of telomerase activity. The oligonucleotides telo-RZ– and telo-RZ+ were annealed and digested with the NotI restriction enzyme. The resulting fragment, with NotI protruding ends, was inserted into the pRc/CMV (Invitrogen, San Diego, CA) mammalian expression vector, previously digested with NotI. The sequence and the correct orientation of the insert in the vector was verified by DNA sequencing, and the resultant vector was named pRcRz. DOTAP-mediated transfection of JR8 cells was performed according to the manufacturer's instructions. Briefly, cells previously seeded at a density yielding approximately 50% confluency at the time of transfection, were transfected with 5 μg of pRcRz vector (or pRc/CMV control vector) that had been complexed with 30 μg of DOTAP. Six hours after transfection, the culture medium containing the DOTAP/DNA mixture was replaced by a selection medium containing G418 to a final concentration of 2 mg per ml. The transfected cells were exposed to G418 for 1 mo. Total RNA was isolated from the transfectants and parental JR8 cell line, and reverse-transcribed as previously described. To analyze ribozyme expression, the resultant cDNA was amplified using T7 and SP6 primers (M-Medical) and by performing 32 cycles of PCR (95°C for 45 s, 48°C for 30 s, and 72°C for 45 s) followed by a 7 min extension step at 72°C in the presence of 1 μCi/sample of [α-32P]deoxycytidine triphosphate (3000 Ci per mmol, Amersham) in a 50 μl of finale volume. For the analysis of hTR expression, reverse transcriptase–mixtures were diluted 10,000 fold. One microliter of each solution was amplified in the presence of teloS and teloA primers and 1 μCi of [α-32P]deoxycytidine triphosphate, by performing 26 cycles of PCR (95°C for 30 s, 65°C for 45 s, and 72°C for 30 s) followed by a 5 min extension step at 72°C, in a final volume of 20 μl. β-Actin, used as a standard of amplification, was coamplified with hTR RNA using two specific primers (774: 5′-GGGAATTCAAAACTGGAA-CGGTGAAGG-3′ 775: 5′-GGAAGCTTATCAAAGTCCTCGGCCA-CA-3′) (Ulaner et al., 1998Ulaner G.A. Hu J.F. Vu T.H. Giudice L.C. Hoffman A.R. Telomerase activity in human development is regulated by human telomerase reverse transcriptase (hTERT) transcription and by alternate splicing of hTERT transcripts.Cancer Res. 1998; 58: 4168-4172PubMed Google Scholar). The PCR products were analyzed by electrophoresis on a 5% nondenaturating polyacrylamide gel in 1 × Tris-borate EDTA buffer (TBE; 45 mM Tris-borate, pH 8.3, 1 mM EDTA). The gel was dried and autoradiographed. Telomerase activity was measured by the telomeric repeat amplification protocol (TRAP) as described byKim et al., 1994Kim N.W. Piatyszek M.A. Prowse K.R. et al.Specific association of human telomerase activity with immortal cells and cancer.Science. 1994; 266: 2011-2015Crossref PubMed Scopus (6308) Google Scholar. Five to 106 cells (or 50–100 mg of melanoma tissues) were resuspendend in ice-cold lysis buffer (10 mM Tris–HCl, pH 7.5, 1 mM MgCl2, 1 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 0.1 mM phenylmethylsulfonyl fluoride, 5 mMβ-mercaptoethanol, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate) and kept on ice for 30 min. After centrifugation at 25,000 × g at 4°C for 30 min, the supernatants were quick-frozen in liquid nitrogen and stored at -80°C. Protein concentrations of the lysates were determined using standard procedures. Proteins (2 μg) from each extract were assayed in 50 μl of TRAP reaction mixture composed of 20 mM Tris–HCl, pH 8.3, 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween-20, 1 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 50 mM each of deoxynucleoside triphosphates, 0.1 μg telomerase substrate (TS) oligonucleotide, 1 μg of T4g32 protein (Boehringer Mannheim), 0.1 mg bovine serum albumin per ml, 2 U Taq DNA polymerase (AmpliTaq, Perkin Elmer), 0.2 μl of [α-32P]deoxycytidine triphosphate (10 μCi per μl, 3000 Ci per mmol, Amersham International). After 20 min of incubation at 23°C for extension of the TS oligonucleotide by telomerase, the reaction mixture was heated at 94°C for 30 s and then subjected to 30 PCR cycles (94°C for 30 s, 50°C for 30 s, 72°C for 45 s), followed by a 3 min extension step at 72°C. The total reaction mixture was analyzed by electrophoresis on a 10% nondenaturating polyacrylamide gel. For each cell extract, aliquots containing 2 μg of protein were pretreated with 20 μg per ml RNase A for 20 min at 37°C and used as a control to verify the specific telomerase activity. For quantitative analysis, assays were repeated using the Oncor TRAPeze Telomerase Detection Kit (Oncor Appligene, Heidelberg, Germany). Each reaction product was amplified in the presence of a 36 bp internal TRAP assay standard (ITAS), and each sample extract was tested for RNase sensitivity. A TSR8 quantitation standard was included for each set of TRAP assays. The TSR8 control is provided in the kit and serves as a standard for estimating the amount of product extended by telomerase in a given extract. Quantitative analysis was performed with the Image-QuanT software (Molecular Dynamics, Sunnyvale, CA), which allowed densitometric evaluation of the digitized image. Telomerase activity was quantitated by measuring the signal of telomerase ladder bands, and the relative telomerase activity was calculated as the ratio to the internal standard using the following formula relativetelomeraseactivity:[(X-X0)/C]×[(R-R0)/Cr]-1 where X is the untreated sample, X0 is the RNase-treated sample, C is the internal control of untreated samples, Cr is the internal control of TSR8, R is the TSR8 quantitation control, and R0 is the negative control. The effect of ribozyme on telomerase was expressed as the percentage inhibition of enzyme activity in samples exposed to the ribozyme compared with controls. The extent of inhibition as a function of the ribozyme concentration was plotted and these graphs were utilized to derive the IC50 values. Total DNA was isolated using DNAzol (Life Technologies, Gaithersburg, MD). For each sample, 10 μg of DNA was digested with 40 units of HinfI and then electrophoresed on 0.8% agarose gels. Following electrophoresis, gels were denatured, neutralized, transferred to a nylon membrane (Hybond N; Amersham), and then cross-linked with ultraviolet light. The membrane was hybridized with a 5′-end [α-32P]deoxyadenosine triphosphate-labeled telomeric oligonucleotide probe (TTAGGG)4. Hybridization was carried out at 42°C for 1 h in rapid hybridization buffer (Amersham). The membrane was washed in 5 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate at room temperature and 1 × sodium citrate/chloride buffer, 0.1% sodium dodecyl sulfate at 42°C. Filters were autoradiographed (Hyperfilm-MP; Amersham) with an intensifying screen at -80°C for 12–24 h. Autoradiographs were scanned (ScanJet IIcx/T; Hewlett Packard) and mean telomere length was calculated as previously reported (Mehle et al., 1994Mehle C. Ljungberg B. Ross G. Telomere shortening in renal carcinoma.Cancer Res. 1994; 54: 236-241PubMed Google Scholar). As the first step of the study, we verified the catalytic potential of the RZH ribozyme on an internally 32P-labeled synthetic RNA substrate obtained by cloning a portion of the RNA component of human telomerase. The size of the resulting substrate was 165 bp. Preliminary experiments performed by incubating 0.1 μM labeled RNA substrate with increasing concentrations (from 0.01 to 1.0 μM) of the ribozyme for 2 h at 37°C resulted in the expected cleavage products of 102 bp and 63 bp, respectively. The efficiency of cleavage was dependent on the ribozyme/substrate ratio, with cleavage products being detectable at a ratio of 0.5:1, and increased with increasing amounts of the ribozyme relative to the substrate (data not shown). In parallel, we assessed the catalytic activity of the RZH ribozyme as a function of MgCl2 concentration. Cleavage was absolutely dependent on Mg2+, but it was already detectable at MgCl2 concentrations as low as 1 mM (data not shown). Based on these results, we carried out a quantitative analysis of RZH ribozyme catalytic activity under experimental conditions thought to guarantee an efficient cleavage reaction (Figure 2). In addition to being concentration-dependent, ribozyme catalytic activity was more efficient at 37°C than at 23°C. Specifically, whereas 95% of the substrate was cleaved in the presence of 10 μM RZH over a 2 h incubation at 37°C, only 78% of substrate cleavage was observed after 12 h of incubation with the same ribozyme concentration at 23°C. A greater cleavage activity of the ribozyme at the higher temperature was also appreciable at the concentration of 1 μM, whereas the same efficiency of cleavage at 23°C and 37°C was evident at the intermediate ribozyme concentration (3 μM). A weak band, corresponding to a fragment of approximately 102 bp, was observed even in lanes containing only the RNA substrate. It may not have been a specific cleavage product but only an artifact produced during the in vitro transcription for RNA substrate synthesis or, alternatively, a product of substrate degradation. No cleavage was detected when the reaction was performed in the presence of 10 μM of the negative control oligomer at either temperatures. The ability of RZH ribozyme to inhibit the activity of telomerase was assessed on cell extracts from two human melanoma cell lines that displayed a comparable basal level of telomerase activity in the TRAP assay. The JR8 cell extract was mixed with increasing concentrations of RZH and incubated for 3, 6, and 12 h at 23°C. Although the ribozyme is more active at 37°C, the 23°C