summary
The knockdown of genes that are over-expressed in cancer, and function in tumor onset and/or progression, is an attractive tool to impair the growth of tumor cells. Synthetic nucleic acids such as antisense oligodeoxynucleotides (AS-ODNs) or small-interfering RNAs (siRNAs) were applied against different tumor-associated transcripts, including the human telomerase reverse transcriptase (hTERT), to inhibit the proliferation of tumor cells and to sensitize them against chemotherapeutic (CT) agents. The efficacy of nucleic acid-based inhibitors was evaluated in vitro by determining the extent of down-regulation of the respective target mRNA and protein expression as well as by extensively investigating growth properties (e.g., viability, proliferation, apoptosis, and cell-cycle distribution) of the affected tumor cells. Methods for a successful down-regulation of hTERT and for the quantitative determination of resulting effects on cellular growth were described herein.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Nakamura, T. M., Morin, G. B., Chapman, K. B., Weinrich, S. L., Andrews, W. H., Lingner, J., Harley, C. B., and Cech, T. R. (1997) Telomerase catalytic subunit homologs from fission yeast and human. Science 277, 955–9.
Sharma, G. G., Gupta, A., Wang, H., Scherthan, H., Dhar, S., Gandhi, V., Iliakis, G., Shay, J. W., Young, C. S., and Pandita, T. K. (2003) hTERT associates with human telomeres and enhances genomic stability and DNA repair. Oncogene 22, 131–46.
Chen, Z., Koeneman, K. S., and Corey, D. R. (2003) Consequences of telomerase inhibition and combination treatments for the proliferation of cancer cells. Cancer Res 63, 5917–25.
Misawa, M., Tauchi, T., Sashida, G., Nakajima, A., Abe, K., Ohyashiki, J. H., and Ohyashiki, K. (2002) Inhibition of human telomerase enhances the effect of chemotherapeutic agents in lung cancer cells. Int J Oncol 21, 1087–92.
Crooke, S. T. (1999) Molecular mechanisms of action of antisense drugs. Biochim Biophys Acta 1489, 31–44.
Agrawal, N., Dasaradhi, P. V., Mohmmed, A., Malhotra, P., Bhatnagar, R. K., and Mukherjee, S. K. (2003) RNA interference: biology, mechanism, and applications. Microbiol Mol Biol Rev 67, 657–85.
Bilanges, B., and Stokoe, D. (2005) Direct comparison of the specificity of gene silencing using antisense oligonucleotides and RNAi. Biochem J 388, 573–83.
Grunweller, A., Wyszko, E., Bieber, B., Jahnel, R., Erdmann, V. A., and Kurreck, J. (2003) Comparison of different antisense strategies in mammalian cells using locked nucleic acids, $2′$-O-methyl RNA, phosphorothioates and small interfering RNA. Nucleic Acids Res 31, 3185–93.
Tsui, P., Rubenstein, M., and Guinan, P. (2005) siRNA is not more effective than a first generation antisense oligonucleotide when directed against EGFR in the treatment of PC-3 prostate cancer. In Vivo 19, 653–6.
Patzel, V., Steidl, U., Kronenwett, R., Haas, R., and Sczakiel, G. (1999) A theoretical approach to select effective antisense oligodeoxyribonucleotides at high statistical probability. Nucleic Acids Res 27, 4328–34.
Kretschmer-Kazemi Far, R., and Sczakiel, G. (2003) The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucleic Acids Res 31, 4417–24.
Axel, D. I., Spyridopoulos, I., Riessen, R., Runge, H., Viebahn, R., and Karsch, K. R. (2000) Toxicity, uptake kinetics and efficacy of new transfection reagents: increase of oligonucleotide uptake. J Vasc Res 37, 221–34; discussion 303–4.
Kraemer, K., Fuessel, S., Schmidt, U., Kotzsch, M., Schwenzer, B., Wirth, M. P., and Meye, A. (2003) Antisense-mediated hTERT inhibition specifically reduces the growth of human bladder cancer cells. Clin Cancer Res 9, 3794–800.
Kraemer, K., Fuessel, S., Kotzsch, M., Ning, S., Schmidt, U., Wirth, M. P., and Meye, A. (2004) Chemosensitization of bladder cancer cell lines by human telomerase reverse transcriptase antisense treatment. J Urol 172, 2023–8.
De Kok, J. B., Schalken, J. A., Aalders, T. W., Ruers, T. J., Willems, H. L., and Swinkels, D. W. (2000) Quantitative measurement of telomerase reverse transcriptase (hTERT) mRNA in urothelial cell carcinomas. Int J Cancer 87, 217–20.
Yajima, T., Yagihashi, A., Kameshima, H., Furuya, D., Kobayashi, D., Hirata, K., and Watanabe, N. (2000) Establishment of quantitative reverse transcription–polymerase chain reaction assays for human telomerase-associated genes. Clin Chim Acta 290, 117–27.
Chen, J., Wu, W., Tahir, S. K., Kroeger, P. E., Rosenberg, S. H., Cowsert, L. M., Bennett, F., Krajewski, S., Krajewska, M., Welsh, K., Reed, J. C., and Ng, S. C. (2000) Down-regulation of survivin by antisense oligonucleotides increases apoptosis, inhibits cytokinesis and anchorage-independent growth. Neoplasia 2, 235–41.
Acknowledgments
We thank all colleagues from our department for their support as well as Dr M. Kotzsch (Institute for Pathology) and Dr B. Schwenzer (Department of Biochemistry) for permanent encouragement. Furthermore, we express our gratitude to the Pinguin foundation for its grant to K.K. and the Robert Pfleger foundation for its grant to A.M. and S.F.
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
Kraemer, K., Fuessel, S., Meye, A. (2007). Telomerase Inhibition by Synthetic Nucleic Acids and Chemosensitization in Human Bladder Cancer Cell Lines. In: Andrews, L.G., Tollefsbol, T.O. (eds) Telomerase Inhibition. Methods in Molecular Biology™, vol 405. Humana Press. https://doi.org/10.1007/978-1-60327-070-0_2
Download citation
DOI: https://doi.org/10.1007/978-1-60327-070-0_2
Publisher Name: Humana Press
Print ISBN: 978-1-58829-683-2
Online ISBN: 978-1-60327-070-0
eBook Packages: Springer Protocols