Inhibition of Telomerase Activity by a Hammerhead Ribozyme Targeting the RNA Component of Telomerase in Human Melanoma Cells Marco Folini, Gennaro Colella,

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Presentation transcript:

Inhibition of Telomerase Activity by a Hammerhead Ribozyme Targeting the RNA Component of Telomerase in Human Melanoma Cells Marco Folini, Gennaro Colella, Raffaella Villa, Susanna Lualdi, Maria Grazia Daidone, Nadia Zaffaroni Journal of Investigative Dermatology Volume 114, Issue 2, Pages 259-267 (February 2000) DOI: 10.1046/j.1523-1747.2000.00870.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Schematic representation of the hammerhead ribozyme (RZH) annealed to the synthetic substrate obtained from the human RNA telomerase template. The cleavage site on the substrate is indicated. Plasmid pRZ was used to synthesize the ribozyme in vitro using T7 RNA polymerase, and the negative control oligomer using SP6 RNA polymerase. Plasmid pRNAtelo was used to synthesize the substrate in vitro by using SP6 polymerase. Journal of Investigative Dermatology 2000 114, 259-267DOI: (10.1046/j.1523-1747.2000.00870.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Cleavage of a 32P-RNA synthetic substrate (165 bp) by RZH ribozyme into two cleavage products (102 and 63 bp). (a) The synthetic 32P-RNA substrate (0.1 μM) was incubated with increasing concentrations of unlabeled ribozyme. The reaction was carried out at 23°C for 12 h (lanes 1–5) or at 37°C for 2 h (lanes 6–10): substrate alone (lanes 1 and 6); 1 μM (lanes 2 and 7), 3 μM (lanes 3 and 8), and 10 μM RZH ribozyme (lanes 4 and 9); 10 μM negative control oligomer (lanes 5 and 10). (b) Quantitation of cleavage products. The extent of cleavage was calculated as the ratio between the amount of densitometric units of cleavage products (102 and 63 bp) and the overall densitometric units obtained for substrate and cleavage products. The weak band of approximately 102 bp present in lanes 1 and 6 (substrate alone) was considered as the background for the 102 bp cleavage product bands. The data represent mean values (±SD) from three independent experiments. Journal of Investigative Dermatology 2000 114, 259-267DOI: (10.1046/j.1523-1747.2000.00870.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Inhibition of telomerase activity by RZH ribozyme in extracts from the JR8 cell line. (a) Two micrograms of telomerase-positive cell extracts were incubated for 3, 6, and 12 h at 23°C in the presence of increasing concentrations (from 1 to 10 μM) of ribozyme or 10 μM of negative control oligomer. (b) Quantitation of ribozyme-mediated telomerase inhibition in extracts from JR8 and M14 cell lines. The effect of ribozyme on telomerase is expressed as the percentage inhibition of enzyme activity compared with control. The data represent mean ± SD) from three independent experiments. R8, TSR8 quantitation standard. Journal of Investigative Dermatology 2000 114, 259-267DOI: (10.1046/j.1523-1747.2000.00870.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Inhibition of telomerase activity by RZH ribozyme on extracts from a human melanoma surgical specimen (ML 1583). (a) Two micrograms of extract were incubated for 6 h at 23°C in the presence of increasing concentrations (from 1 to 10 μM) of ribozyme or 10 μM of negative control oligomer. (b) Quantitation of RZH ribozyme-mediated telomerase inhibition in cell extracts from three human melanoma surgical specimens. The data represent mean values (±SD) from three independent experiments for each tumor. The effect of ribozyme on telomerase is expressed as the percentage inhibition of enzyme activity compared with control. Journal of Investigative Dermatology 2000 114, 259-267DOI: (10.1046/j.1523-1747.2000.00870.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Inhibition of telomerase activity by RZH ribozyme delivered to JR8 cells through cationic liposome-mediated transfer. Cells exposed to the complex made of 60 μg per 2 ml of DOTAP and 45 μg per 2 ml of negative control oligomer (lane 1); untreated control cells (lane 2); cells exposed to the complex made of 60 μg per 2 ml of DOTAP and 45 μg per 2 ml of RZH ribozyme for 48 h (lane 3); cells exposed to 60 μg per 2 ml DOTAP for 48 h (lane 4). Journal of Investigative Dermatology 2000 114, 259-267DOI: (10.1046/j.1523-1747.2000.00870.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Telomerase activity in transfectants and parental cells. Lane 1, JR8 parental cells; lane 2, JR8 cell extract pretreated with 20 μg per ml RNase; lane 3, lysis buffer; lanes 4–11, ribozyme transfectant clones 1–8; lane 12, pooled clone of ribozyme transfectants; lane 13, vector transfectant clone. Journal of Investigative Dermatology 2000 114, 259-267DOI: (10.1046/j.1523-1747.2000.00870.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Ribozyme and telomerase RNA expression as detected by reverse transcriptase–PCR in transfectants and parental cells. The ribozyme was expressed and the telomerase RNA level was diminished in all ribozyme transfectant clones. β-Actin was used as a control for amplification. Lane 1, JR8 parental cells; lane 2, vector transfectant clone; lanes 3–5, ribozyme transfectant clones, lane 6, pooled clone of ribozyme transfectants. Journal of Investigative Dermatology 2000 114, 259-267DOI: (10.1046/j.1523-1747.2000.00870.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Morphology of ribozyme transfectants and parental cells. (a) JR8 parental cells. (b) Ribozyme transfectant cells (clone 2). Cells were grown for 3 d in monolayer culture. Scale bar: 30 μm. Journal of Investigative Dermatology 2000 114, 259-267DOI: (10.1046/j.1523-1747.2000.00870.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Telomere length of ribozyme transfectants and parental cells. A similar telomere length, as detected by southern blot hybridization, was observed for JR8 parental cells and ribozyme transfectants (clones 1, 2, 7, and pooled clone) after 30 doublings in culture. Journal of Investigative Dermatology 2000 114, 259-267DOI: (10.1046/j.1523-1747.2000.00870.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions