Abstract
In recent years expanding knowledge about basic biology and a detailed understanding of the molecular pathways involved in tumor cell growth and progression have allowed the identification of numerous genes as potential therapeutic targets. Studies in which the expression of these genes was manipulated by antisense strategies have provided clues as to how we can intervene to specifically kill tumor cells or sensitize them to conventional chemical and physical antitumor therapies. Such tumor specificity can only be obtained by exploiting a basic difference between normal and malignant cells. In this context, targeting cytoprotective factors such as telomerase and survivin is particularly attractive because of their almost selective expression in tumor cells and their proven association with disease progression. This chapter summarizes the results obtained with ribozymes and small-interfering RNAs in the functional validation of these two targets in cell cultures and animal tumor models.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jansen, B. and Zangemeister-Wittke, U. (2002) Antisense therapy for cancer—the time of truth. Lancet Oncol. 3, 672–683.
Kyo, S. and Inoue, M. (2002) Complex regulatory mechanisms of telomerase activity in normal and cancer cells: how can we apply them for cancer therapy? Oncogene 21, 688–697.
Altieri, D. C. (2003) Survivin and apoptosis control. Adv. Cancer Res. 88, 31–52.
Hahn, W. C. (2003) Role of telomeres and telomerase in the pathogenesis of human cancer. J. Clin. Oncol. 21, 2034–2043.
Smogorzewska, A. and de Lange, T. (2004) Regulation of telomerase by telomeric proteins. Annu. Rev. Biochem. 73, 177–208.
Karlseder, J. (2003) Telomere repeat binding factor: keeping the ends in check. Cancer Lett. 194, 189–197.
Wright, W. E. and Shay, J. W. (2005) Telomere-binding factors and general DNA repair. Nat. Genet. 37, 116–118.
Smogorzewska, A., Karlseder, J., Holtgreve-Grez, H., Jauch, A., and de Lange, T. (2002) DNA ligase IV-dependent NHEJ of deprotected mammalian telomeres in G1 and G2. Curr. Biol. 12, 1635–1644.
Baumann, P. and Cech, T. R. (2001) Pot1, the putative telomere end-binding protein in fission yeast and humans. Science 292, 1171–1175.
Lundblad, V. (2003) Telomeres: taking the measure. Nature 423, 926–927.
Colgin, L. M., Baran, K., Baumann, P., Cech, T. R., and Reddel, R. R. (2003) Human POT1 facilitates telomere elongation by telomerase. Curr. Biol. 13, 942–946.
Keith, W. N., Evans, T. R. J., and Glasspool, R. M. (2001) Telomerase and cancer: time to move from a promising target to a clinical reality. J. Pathol. 195, 404–414.
Olovnikov, A. M. (1973) A theory of marginotomy. The incomplete copy of template margin in enzymatic synthesis of polynucleotides and biological significance of the phenomenon. J. Theor. Biol. 41, 181–190.
Cong, Y. S., Wright, W. E., and Shay, J. W. (2002) Human telomerase and its regulation. Microbiol. Mol. Biol. Rev. 66, 407–425.
Harrington, L., Zhou, W., McPhail, T., et al. (1997) Human telomerase contains evolutionarily conserved catalytic and structural subunits. Genes Dev. 11, 3109–3115.
Feng, J., Funk, W. D., Wang, S. S., et al. (1995) The RNA component of human telomerase. Science 269, 1236–1241.
Yi, X., White, D. M., Aisner, D. L., Baur, J. A., Wright, W. E., and Shay, J. W. (2000) An alternate splicing variant of the human telomerase catalytic subunit inhibits telomerase activity. Neoplasia 2, 433–440.
Blackburn, E. H. (2005) Telomeres and telomerase: their mechanisms of action and the effects of altering their functions. FEBS Lett. 579, 859–862.
Hahn, W. C. and Meyerson, M. (2001) Telomerase activation, cellular immortalization and cancer. Ann. Med. 2, 123–129.
Shay, J. W. and Bacchetti, S. (1997) A survey of telomerase activity in human cancer. Eur. J. Cancer 33, 787–791.
Hanahan, D. and Weinberg, R. A. (2000) The hallmarks of cancer. Cell 100, 57–70.
Folini, M. and Zaffaroni, N. (2005) Targeting telomerase by antisense-based approaches: perspectives for new anti-cancer therapies. Curr. Pharm. Des. 11, 1105–1117.
Blackburn, E. H. (2000) Telomere states and cell fates. Nature 408, 53–56.
Sharma, G. G., Gupta, A., Wang, H., et al. (2003) hTERT associates with human telomeres and enhances genomic stability and DNA repair. Oncogene 22, 131–146.
Cao, Y., Li, H., Deb, S., and Liu, J. P. (2004) TERT regulates cell survival independent of telomerase enzymatic activity. Oncogene 21, 3130–3138.
Kelland, L. R. (2005) Overcoming the immortality of tumor cells by telomere and telomerase based cancer therapeutics—current status and future prospects. Eur. J. Cancer 41, 971–979.
Altieri, D. (2004) Molecular circuits of apoptosis regulation and cell division control: the survivin paradigm. J. Cell. Biochem. 92, 656–663.
Ambrosini, G., Adida, C., Sirugo, A., and Altieri, D. C. (1998) Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J. Biol. Chem. 273, 11,177–11,182.
Ambrosini, G., Adida, C., and Altieri, D. C. (1997) A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat. Med. 3, 917–921.
LaCasse, E. C., Baird, S., Korneluk, R. G., and MacKenzie, A. E. (1998) The inhibitors of apoptosis (IAPs) and their emerging role in cancer. Oncogene 17, 3247–3259.
Mahotka, C., Wenzel, M., Springer, E., Gabbert, H. E., and Gerharz, C. D. (1999) Survivin-ΔEx3 and survivin-2B: two novel splice variants of the apoptosis inhibitor survivin with different antiapoptotic properties. Cancer Res. 59, 6097–6102.
Badran, A., Yoshida, A., Ishikawa, K., et al. (2004) Identification of a novel splice variant of the human anti-apoptopsis gene survivin Biochem. Biophys. Res. Commun. 314, 902–907.
Caldas, H., Honsey, L. E., and Altura, R. A. (2005) Survivin 2alpha: a novel Survivin splice variant expressed in human malignancies. Mol. Cancer 4, 11.
Caldas, H., Jiang, Y., Holloway, M. P., et al. (2005) Survivin splice variants regulate the balance between proliferation and cell death. Oncogene 24, 1994–2007.
Li, F. and Altieri, D. C. (1999) The cancer anti-apoptosis mouse survivin gene: characterization of locus and transcriptional requirements of basal and cell cycle-dependent expression. Cancer Res. 59, 3143–3151.
O’Connor, D. S., Grossman, D., Plescia, J., et al. (2000) Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc. Natl. Acad. Sci. 97, 13,103–13,107.
Fortugno, P., Wall, N. R., Giodini, A., et al. (2002) Survivin exists in immunochemically distinct subcellular pools and is involved in spindle microtubule function. J. Cell Sci. 115, 575–585.
Uren, A. G., Wong, L., Pakusch, M., et al. (2000) Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype. Curr. Biol. 10, 1319–1328.
Li, F., Ackermann, E. J., Bennett, C. F., et al. (1999) Pleiotropic cell-division defects and apoptosis induced by interference with survivin function. Nat. Cell Biol. 1, 461–466.
Chen, J., Wu, W., Tahir, S. K., et al. (2000) Down-regulation of survivin by antisense oligonucleotides increases apoptosis, inhibits cytokinesis and anchorage-dependent growth. Neoplasia 2, 235–241.
Giodini, A., Kallio, M. J., Wall, N. R., et al. (2002) Regulation of microtubule stability and mitotic progression by survivin. Cancer Res. 62, 2462–2467.
Kallio, M. J., Nieminen, M., and Eriksson, J. E. (2001) Human inhibitor of apoptosis protein (IAP) survivin participates in regulation of chromosome segregation and mitotic exit. FASEB 15, 2721–2723.
Carvalho, A., Carmena, M., Sambade, C., Earnshaw, W. C., and Wheatley, S. P. (2003) Survivin is required for stable checkpoint activation in taxol-treated HeLa cells. J. Cell. Sci. 116, 2987–2998.
Dohi, T., Beltrami, E., Wall, N. R., Plescia, J., and Altieri, D. C. (2004) Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis. J. Clin. Invest. 114, 1117–1127.
Fortugno, P., Beltrami, E., Plescia, J., et al. (2003) Regulation of survivin function by Hsp90. Proc. Natl. Acad. Sci. USA 100, 13,791–13,796.
O’Connor, D. S., Grossman, D., Plescia, J., et al. (2000) Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc. Natl. Acad. Sci. 97, 13,103–13,107.
Song, Z., Yao, X., and Wu, M. (2003) Direct interaction between survivin and Smac/DIABLO is essential for the anti-apoptotic activity of survivin during taxolinduced apoptosis. J. Biol. Chem. 278, 23,130–23,140.
Shiozaki, E. N. and Shi, Y. (2004) Caspases, IAPs and Smac/DIABLO: mechanisms from structural biology. Trends Biochem. Sci. 29, 486–494.
Altieri, D. C. (2003) Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene 22, 8581–8589.
O’Connor, D. S., Schechner, J. S., Adida, C., et al. (2000) Control of apoptosis during angiogenesis by survivin expression in endothelial cells. Am. J. Pathol. 156, 393–398.
Velculescu, V. E., Madden, S. L., Zhang, L., et al. (1999) Analysis of human transcriptomes. Nat. Gen. 23, 387–388.
Plantaz, D., Mohapatra, G., Matthay, K. K., Pellarin, M., Seeger, R. C., and Feuerstein, B. G. (1997) Gain of chromosome 17 is the most frequent abnormality detected in neuroblastoma by comparative genomic hybridization. Am. J. Pathol. 150, 81–89.
Hattori, M., Sakamoto, H., Satoh, K., and Yamamoto, T. (2001) DNA demethylase is expressed in ovarian cancers and the expression correlates with demethylation of CpG sites in the promoter region of c-erbB-2 and survivin genes. Cancer Lett. 169, 155–164.
Mirza, A., McGuirk, M., Hockenberry, T. N., et al. (2002) Human survivin is negatively regulated by wild-type p53 and participates in p53-dependent apoptotic pathway. Oncogene 21, 2613–2622.
Li, F., Ambrosini, G., Chu, E. Y., et al. (1998) Control of apoptosis and mitotic spindle checkpoint by survivin. Nature 396, 580–584.
Zaffaroni, N., Pennati, M., Colella, G., et al. (2002) Expression of the antiapoptotic gene survivin correlates with taxol resistance in human ovarian cancer. Cell. Mol. Life Sci. 59, 1406–1412.
Asanuma, K., Moriai, R., Yajima, T., et al. (2000) Survivin as a radio-resistance factor in pancreatic cancer. Jap. J. Cancer Res. 91, 1204–1209.
Zhang, M., Latham, D. E., Delaney, M. A., and Chakravarti, A. (2005) Survivin mediates resistance to antiandrogen therapy in prostate cancer. Oncogene 24, 2474–2482.
Nicholson, D. W. (2000) From bench to clinic with apoptosis-based therapeutic agents. Nature 407, 810–816.
Fischer, U. and Schulze-Osthoff, K. (2005) New approaches and therapeutics targeting apoptosis in disease. Pharmacol. Rev. 57, 187–215.
Altieri, D. C. (2003) Validating survivin as a cancer therapeutic target. Nat. Rev. Cancer 3, 46–54.
Puerta-Fernandez, E., Romer-Lopez, C., Barroso-delJesus, A., and Berzal-Herranz, A. (2003) Ribozymes: recent advances in the development of RNA tools. FEMS Microbiol. Rev. 27, 75–97.
Kore, A. R., Vaish, N. K., Kutzke, U., and Eckstein, F. (1998) Sequence specificity of the hammerhead ribozyme revisited: the NHH rule. Nucleic Acids Res. 26, 4116–4120.
Kurreck, J. (2003) Antisense technologies. Improvement through novel chemical modifications. Eur. J. Biochem. 270, 1628–1644.
Kanazawa, Y., Ohkawa, K., Ueda, K., et al. (1996) Hammerhead ribozyme-mediated inhibition of telomerase activity in extracts of human hepatocellular carcinoma cells. Biochem. Biophys. Res Commun. 225, 570–576.
Wan, M. S., Fell, P. L., and Akhtar, S. (1998) Synthetic 2′-O-methyl-modified hammerhead ribozymes targeted to the RNA component of telomerase as sequence-specific inhibitors of telomerase activity. Antisense Nucleic Acid Drug Dev. 8, 309–317.
Folini, M., Colella, G., Villa, R., Lualdi, S., Daidone, M. G., and Zaffaroni, N. (2000) Inhibition of telomerase activity by a hammerhead ribozyme targeting the RNA component of telomerase in human melanoma cells. J. Invest. Dermatol. 114, 259–267.
Yokoyama, Y., Takahashi, Y., Shinohara, A., et al. (1998) Attenuation of telomerase activity by a hammerhead ribozyme targeting the template region of telomerase RNA in endometrial carcinoma cells. Cancer Res. 58, 5406–5410.
Yokoyama, Y., Wan, X., Takahashi, Y., Shinohara, A., Liulin, T., and Tamaya, T. (2002) Divalent hammerhead ribozyme targeting template region of human telomerase RNA has potent cleavage activity, but less inhibitory activity on telomerase. Arch. Biochem. Biophys. 405, 32–37.
Yeo, M., Rha, S. Y., Jeung, H. C., et al. (2005) Attenuation of telomerase activity by hammerhead ribozyme targeting human telomerase RNA induces growth retardation and apoptosis in human breast tumor cells. Int. J. Cancer 114, 484–489.
Yokoyama, Y., Takahashi, Y., Shinohara, A., et al. (2000) The 5′-end of hTERT mRNA is a good target for hammerhead ribozyme to suppress telomerase activity. Biochem. Biophys. Res Commun. 273, 316–321.
Ludwig, A., Saretzki, G., Holm, P. S., et al. (2001) Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase. Cancer Res. 61, 3053–3061.
Saretzki, G., Ludwig, A., von Zglinicki, T., and Runnebaum, I. B. (2001) Ribozyme-mediated telomerase inhibition induces immediate cell loss but not telomere shortening in ovarian cancer cells. Cancer Gene Ther. 8, 827–834.
Pennati, M., Colella, G., Folini, M., Citti, L., Daidone, M. G., and Zaffaroni, N. (2002) Ribozyme-mediated attenuation of survivin expression sensitizes human melanoma cells to cisplatin-induced apoptosis. J. Clin. Invest. 109, 285–286.
Pennati, M., Binda, M., De Cesare, M., et al. (2004) Ribozyme-mediated down-regulation of survivin expression sensitizes human melanoma cells to topotecan in vitro and in vivo. Carcinogenesis 25, 1129–1136.
Pennati, M., Binda, M., Coltella, G., et al. (2003) Radiosensitization of human melanoma cells by ribozyme-mediated inhibition of survivin expression. J. Invest. Dermatol. 120, 648–654.
Pennati, M., Binda, M., Coltella, G., et al. (2004) Ribozyme-mediated inhibition of survivin expression increases spontaneous and drug-induced apoptosis and decreases the tumorigenic potential of human prostate cancer cells. Oncogene 23, 386–394.
Choi, K. S., Lee, T. H., and Jung, M. H. (2003) Ribozyme-mediated cleavage of the human survivin mRNA and inhibition of antiapoptotic function of survivin in MCF-7 cells. Cancer Gene Ther. 10, 87–95.
Elbashir, S. M., Harborth, J., Weber, K., and Tuschl, T. (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26, 199–213.
Dykxhoorn, D. M., Novina, C. D., and Sharp, P. A. (2003) Killing the messenger: short RNAs that silence gene expression. Nat. Rev. Mol. Cell. Biol. 4, 457–467.
Izquierdo, M. (2005) Short interfering RNAs as a tool for cancer gene therapy. Cancer Gene Ther. 12, 217–227.
Kosciolek, B. A., Kalantidis, K., Tabler, M., and Rowley, P. T. (2003) Inhibition of telomerase activity in human cancer cells by RNA interference. Mol. Cancer Ther. 2, 209–216.
Li, S., Crothers, J., Haqq, C. M., and Blackburn, E. H. (2005) Cellular and gene expression responses involved in the rapid growth inhibition of human cancer cells by RNA interference-mediated depletion of telomerase RNA. J. Biol. Chem. 280, 23,709–23,717.
Kappler, M., Bache, M., Bartel, F., et al. (2004) Knockdown of survivin expression by small interfering RNA reduces the clonogenic survival of human sarcoma cell lines independently of p53. Cancer Gene Ther. 11, 186–193.
Kappler, M., Taubert, H., Bartel, F., et al. (2005) Radiosensitization, after a combined treatment of survivin siRNA and irradiation, is correlated with the activation of caspases 3 and 7 in a wt-p53 sarcoma cell line, but not in a mt-p53 sarcoma cell line. Oncol. Rep. 13, 167–172.
Chawla-Sarkar, M., Bae, S. I., Reu, F. J., Jacobs, B. S., Lindner, D. J., and Borden, E. C. (2004) Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis. Cell Death Differ. 11, 915–923.
Coma, S., Noe, V., Lavarino, C., et al. (2004) Use of siRNAs and antisense oligonucleotides against survivin RNA to inhibit steps leading to tumor angiogenesis. Oligonucleotides 14, 100–113.
Coumoul, X., Li, W., Wang, R. H., and Deng, C. (2004) Inducible suppression of Fgfr2 and Survivin in ES cells using a combination of the RNA interference (RNAi) and the Cre-LoxP system. Nucleic Acids Res. 32, e85.
Scherer, L. J. and Rossi, J. J. (2003) Approaches for the sequence-specific knockdown of mRNA. Nature Biotechnol. 21, 1457–1465.
Caplen, N. J. (2004) Gene therapy progress and prospects. Downregulating gene expression: the impact of RNA interference. Gene Ther. 11, 1241–1248.
Schubert, S., Grünweller, A., Erdmann, V. A., and Kurreck, J. (2005) Local RNA target structure influences siRNA efficacy: systematic analysis of intentionally designed binding regions. J. Mol. Biol. 348, 883–893.
Overhoff, M., Alken, M., Far, R. K., et al. (2005) Local RNA target structure influences siRNA efficacy: a systematic global analysis. J. Mol. Biol. 348, 871–881.
Lee, N. S., Lee, N. S., Bertrand, E., and Rossi, J. (1999) mRNA localizasion signals can enhance the intracellular effectiveness of hammerhead ribozymes. RNA 5, 1200–1209.
Sullenger, B. A. and Gilboa, E. (2002) Emerging clinical application of RNA. Nature 418, 252–258.
Bantounas, I., Phylactou, L. A., and Uney, J. B. (2004) RNA interference and the use of small interfering RNA to study gene function in mammalian systems. J. Mol. Endocrinol. 33, 545–557.
Sledz, C. A. and Williams, B. R. G. (2004) RNA interference and double-stranded-RNA-activated pathways. Biochem. Soc. Trans. 32, 952–956.
Sledz, C. A., Holko, M., de Veer, M. J., Silverman, R. H., and Williams, R. G. (2003) Activation of interferon system by short-interfering RNAs. Nat. Cell. Biol. 5, 834–839.
Huppi, K., Martin, S. E., and Caplen, N. J. (2005) Defining and assaying RNAi in mammalian cells. Mol. Cell. 17, 1–10.
Shay, J. W. and Wright, W. E. (2002) Telomerase: a target for cancer therapeutics. Cancer Cell 2, 257–265.
Folini, M., Brambilla, C., Villa, R., et al. (2005) Antisense oligonucleotide-mediated inhibition of hTERT, but not hTERC, induces rapid cell growth decline and apoptosis in the absence of telomere shortening in human prostate cancer cells. Eur. J. Cancer 41, 624–634.
Henson, J. D., Neumann, A. A., Yeager, T. R., and Reddel, R. R. (2002) Alternative lengthening of telomeres in mammalian cells. Oncogene 21, 598–610.
Reddel, R. R. and Bryan, T. M. (2003) Alternative lengthening of telomeres: dangerous road less travelled. Lancet 361, 1840.
Tran, J., Master, Z., Yu, J. L., Rak, J., Dumont, D. J., and Kerbel, R. S. (2002). A role for survivin in chemoresistance of endothelial cells mediated by VEGF. Proc. Natl. Acad. Sci. USA 99, 4349–4354.
Mesri, M., Morales-Ruiz, M., Ackermann, E. J., et al. (2001) Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting. Am. J. Pathol. 158, 1757–1765.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Zaffaroni, N., Pennati, M., Folini, M. (2007). Validation of Telomerase and Survivin as Anticancer Therapeutic Targets Using Ribozymes and Small-Interfering RNAs. In: Sioud, M. (eds) Target Discovery and Validation Reviews and Protocols. Methods in Molecular Biology™, vol 361. Humana Press. https://doi.org/10.1385/1-59745-208-4:239
Download citation
DOI: https://doi.org/10.1385/1-59745-208-4:239
Publisher Name: Humana Press
Print ISBN: 978-1-58829-890-4
Online ISBN: 978-1-59745-208-3
eBook Packages: Springer Protocols