Summary
New genes useful in suicide gene therapy are those encoding toxins such as plant ribosome-inactivating proteins (RIPs), which can irreversibly block protein synthesis, triggering apoptotic cell death. Plasmids expressing a cytosolic saporin (SAP) gene from common soapwort (Saponaria officinalis) are generated by placing the region encoding the mature plant toxin under the control of strong viral promoters and may be placed under tumor-specific promoters. The ability of the resulting constructs to inhibit protein synthesis is tested in cultured tumor cells co-transfected with a luciferase reporter gene. SAP expression driven by the cytomegalovirus (CMV) promoter (pCI-SAP) demonstrates that only 10 ng of plasmid DNA per 1.6 × 104 B16 melanoma cells drastically reduces luciferase reporter activity to 18% of that in control cells (1). Direct intratumoral injections are performed in an aggressive melanoma model. B16 melanoma-bearing mice injected with pCI-SAP complexed with lipofectamine or N-(2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate (DOTAP) show a noteworthy attenuation in tumor growth, and this effect is significantly augmented by repeated administrations of the DNA complexes. Here, we describe in detail this cost-effective and safe suicide gene approach.
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References
Zarovni, N., Vago, R., Soldà, T., Monaco, L., and Fabbrini, M. S. (2007) Saporin as a novel suicide gene in anticancer gene therapy. Cancer Gene Ther. 14, 165–73.
Martin, V., Cortes, M. L., de Felipe, P., Farsetti, A., Calcaterra, N. B., and Izquierdo, M. (2000) Cancer gene therapy by thyroid hormone-mediated expression of toxin genes. Cancer Res 60, 3218–24.
Yerushalmi, N., Brinkmann, U., Brinkmann, E., Pai, L., and Pastan, I. (2000) Attenuating the growth of tumors by intratumoral administration of DNA encoding Pseudomonas exotoxin via cationic liposomes. Cancer Gene Ther 7, 91–6.
Anderson, D. G., Peng, W., Akinc, A., Hossain, N., Kohn, A., Padera, R., Langer, R., and Sawicki, J. A. (2004) A polymer library approach to suicide gene therapy for cancer Proc Natl Acad Sci USA 101, 16028–33.
Hartley, M. and Lord, J. (2004) Cytotoxic ribosome-inactivating lectins from plants. Biochim Biophys Acta 1701, 1–14.
Sandvig, K. and van Deurs, B. (2002) Transport of protein toxins into cells: pathways used by ricin, cholera toxin and Shiga toxin. FEBS Lett 529, 49–53.
Endo, Y., Mitsui, K., Motizuki, M., and Tsurugi, K. (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem 262, 5908–12.
Bagga, S., Seth, D., and Batra, J. K. (2003) The cytotoxic activity of ribosome-inactivating protein saporin-6 is attributed to its rRNA N-glycosidase and internucleosomal DNA fragmentation activities. J Biol Chem 278, 4813–20.
Hoganson, D. K., Chandler, L. A., Fleurbaaij, G. A., Ying, W., Black, M. E., Doukas, J., Pierce, G. F., Baird, A., and Sosnowski, B. A. (1998) Targeted delivery of DNA encoding cytotoxic proteins through high-affinity fibroblast growth factor receptors. Hum Gene Ther 9, 2565–75.
Goergen, J. L. and Monaco, L. (2004) Generation of high-recombinant-protein-producing Chinese hamster ovary (CHO) cells. Methods Mol Biol 267, 477–83.
Monaco, L., Tagliabue, R., Soria, M. R., and Uhlen, M. (1994) An in vitro amplification approach for the expression of recombinant proteins in mammalian cells. Biotechnol Appl Biochem 20, 157–71.
Fabbrini, M. S., Rappocciolo, E., Carpani, D., Solinas, M., Valsasina, B., Breme, U., Cavallaro, U., Nykjaer, A., Rovida, E., Legname, G., and Soria, M. R. (1997) Characterization of a saporin isoform with lower ribosome-inhibiting activity. Biochem J 322, 719–27.
Majoul, I. V., Bastiaens, P. I., and Soling, H. D. Transport of an External Lys-Asp-Glu-Leu (KDEL) (1996) Protein from the Plasma Membrane to the Endoplasmic Reticulum-Studies with Cholera Toxin in Vero Cells. J Cell Biol 133, 777–89.
Wales, R., Roberts, L. M., and Lord, J. M. (1993) Addition of an endoplasmic reticulum retrieval sequence to ricin A chain significantly increases its cytotoxicity to mammalian cells. J Biol Chem 268, 23986–90.
Vago, R., Marsden, C. J., Lord, J. M., Ippoliti, R., Flavell, D. J., Flavell, S. U., Ceriotti, A., and Fabbrini, M. S. (2005) Saporin and ricin A chain follow different intracellular routes to enter the cytosol of intoxicated cells FEBS J 272, 4983–95.
Zhang, S., Xu, Y., Wang, B., Qiao, W., Liu, D., and Li, Z. (2004) Cationic compounds used in lipoplexes and polyplexes for gene delivery. J Control Rel 100, 165–80.
Gebhart, C. L. and Kabanov, A. V. (2001) Evaluation of polyplexes as gene transfer agents. J Control Rel 73, 401–16.
Missol, E., Sochanik, A., and Szala, S. (1995) Introduction of murine IL-4 gene into B16 (F10) melanoma tumors by direct gene transfer with DNA liposome complexes. Cancer Lett 97, 189–93.
Papaioannou, V. and Fox, J. (1993) Efficacy of tribromethanol anesthesia in mice laboratory. Anim Sci 43, 189–92.
Acknowledgments
The authors are deeply indebted to Lucia Monaco for the development of the nonviral vector strategy, the pCISfiLuc reporter gene construct, and especially for her advice and continuous support. This work has been partially supported by the Italian Cancer Research Association (AIRC, grant 131/01) and by IBBA-CNR, Milano.
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Zarovni, N., Vago, R., Fabbrini, M.S. (2009). Saporin Suicide Gene Therapy. In: Walther, W., Stein, U. (eds) Gene Therapy of Cancer. Methods in Molecular Biology™, vol 542. Humana Press. https://doi.org/10.1007/978-1-59745-561-9_14
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DOI: https://doi.org/10.1007/978-1-59745-561-9_14
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