Abstract
Efficient manipulation of Hedgehog (HH)/GLI signaling activity is crucial to the analysis of molecular events underlying HH/GLI-regulated cell fate determination and tumor growth. In this article, we describe the use of retroviral expression systems as a valuable tool to activate or repress Hh-pathway activity in a broad spectrum of mammalian cells—including human cells—either by forced expression of the major Hedgehog-effectors GLI1 and GLI2 or by expression of the short-hairpin RNAs-targeting GLI mRNAs. We focus on two distinct retroviral systems that allow efficient and sustainable expression of GLI proteins in primary cells and cell lines of human origin: (i) a Moloney Murine Leukemia Virus-based and (ii) an HIV-derived lentivirus expression system, which allows transduction of both dividing and quiescent cells.
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References
Ruiz, I. A. A., Palma, V., and Dahmane, N. (2002) Hedgehog-Gli signalling and the growth of the brain. Nat. Rev. Neurosci. 3, 24–33.
Ingham, P. W. and McMahon, A. P. (2001) Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 15, 3059–3087.
McMahon, A. P., Ingham, P. W., and Tabin, C. J. (2003) Developmental roles and clinical significance of hedgehog signaling. Curr. Top Dev. Biol. 53, 1–114.
Berman, D. M., Karhadkar, S. S., Hallahan, A. R., et al. (2002) Medulloblastoma growth inhibition by hedgehog pathway blockade. Science 297, 1559–1561.
Berman, D. M., Karhadkar, S. S., Maitra, A., et al. (2003) Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature 425, 846–851.
Karhadkar, S. S., Bova, G. S., Abdallah, N., et al. (2004) Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature 431, 707–712.
Watkins, D. N., Berman, D. M., Burkholder, S. G., Wang, B., Beachy, P. A. and Baylin, S. B. (2003) Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature 422, 313–317.
Romer, J. T., Kimura, H., Magdaleno, S., et al. (2004) Suppression of the Shh pathway using a small molecule inhibitor eliminates medulloblastoma in Ptc1 (+/−)p53(−/−) mice. Cancer Cell 6, 229–240.
Sanchez, P., Hernandez, A. M., Stecca, B., et al. (2004) Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling. Proc. Nat. Sci. Acad. USA 101, 12,561–12,566.
Thayer, S. P., di Magliano, M. P., Heiser, P. W., et al. (2003) Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425, 851–856.
Kinsella, T. M. and Nolan, G. P. (1996) Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7, 1405–1413.
Heemskerk, M. H., Blom, B., Nolan, G., et al. (1997) Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3. J. Exp. Med. 186, 1597–1602.
Wiznerowicz, M. and Trono, D. (2003) Conditional suppression of cellular genes: lentivirus vector-mediated drug-inducible RNA interference. J. Virol. 77, 8957–8961.
Rubinson, D. A., Dillon, C. P., Kwiatkowski, A. V., et al. (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33, 401–406.
Deng, H., Lin, Q., and Khavari, P. A. (1997) Sustainable cutaneous gene delivery. Nat. Biotechnol. 15, 1388–1391.
Dull, T., Zufferey, R., Kelly, M., et al. (1998) A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463–8471.
Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., and Trono, D. (1997) Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15, 871–875.
Kinzler, K. W., Ruppert, J. M., Bigner, S. H., and Vogelstein, B. (1988) The GLI gene is a member of the Kruppel family of zinc finger proteins. Nature 332, 371–374.
Tanimura, A., Dan, S., and Yoshida, M. (1998) Cloning of novel isoforms of the human Gli2 oncogene and their activities to enhance tax-dependent transcription of the human T-cell leukemia virus type 1 genome. J. Virol. 72, 3958–3964.
Regl, G., Neill, G. W., Eichberger, T., et al. (2002) Human GLI2 and GLI1 are part of a positive feedback mechanism in Basal Cell Carcinoma. Oncogene 21, 5529–5539.
Davis, H. E., Rosinski, M., Morgan, J. R., and Yarmush, M. L. (2004) Charged polymers modulate retrovirus transduction via membrane charge neutralization and virus aggregation. Biophys. J. 86, 1234–1242.
Boukamp, P. Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., and Fusenig, N. E. (1988) Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 761–771.
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Kasper, M., Regl, G., Eichberger, T., Frischauf, AM., Aberger, F. (2007). Efficient Manipulation of Hedgehog/GLI Signaling Using Retroviral Expression Systems. In: Horabin, J.I. (eds) Hedgehog Signaling Protocols. Methods Inmolecular Biology™, vol 397. Humana Press. https://doi.org/10.1007/978-1-59745-516-9_6
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DOI: https://doi.org/10.1007/978-1-59745-516-9_6
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