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Chemical-Based Translational Induction of Luciferase Expression: An Efficient Tool for in Vivo Screening of Protein Farnesylation Inhibitors

Institut National de la Santé et de la Recherche Médicale U563, Centre de Physiopathologie Toulouse Purpan, Département Innovation Thérapeutique et Oncologie Moléculaire, Toulouse, France (O.B., J.C.F.); Institut Claudius Regaud Toulouse, Université Paul Sabatier, Toulouse, France (C.G., G.F.); Institut National de la Santé et de la Recherche Médicale U 589 ILB, Centre Hospitalier Universitaire Rangueil, Toulouse, France (C.B., S.V.); and MilleGen, Prologue Biotech, Labège, France

We describe the development of a cell system for in vivo screening of inhibitors of the mevalonate pathway. To this aim, we have constructed a bicistronic mRNA, transcribed from a constitutive cytomegalovirus promoter, containing the Renilla reniformis luciferase RNA open reading frame sequence as first cistron and the Firefly luciferase RNA sequence as a second cistron. The intercistronic space is made of the R17 binding sequence of the bacteriophage R17 protein. A chimeric protein able to bind to a specific sequence in the hairpin and to induce internal ribosome entry in the RNA switches on translation of the second cistron. This chimeric protein is made up of the bacteriophage RNA binding domain (R17) fused to the ribosome recruitment core of the eIF-4G1 eukaryotic translation initiation factor and to the CAAX box of H-Ras addressing the protein to the plasma membrane where it is not efficient. Internal ribosome entry upstream of the Firefly cistron is therefore under the dependence of the mevalonate pathway inhibitors. Indeed, products that are able to inhibit protein farnesylation rescue the cytoplasmic location of the R17-eIF-4G-CAAX protein, which once more becomes a translation factor for the expression of the second cistron. To exemplify the system, the present work checks the ability of various antiestrogens to interfere with the mevalonate pathway. It seems that pure antiestrogen, able to selectively bind the estrogen receptor, is unable to switch on the second Firefly cistron although selective antiestrogen-binding-site ligands are able to do so.

Received for publication January 13, 2005.

Accepted for publication March 22, 2005.

Address correspondence to: Jean-Charles Faye, INSERM U563, ICR, 20–24 Rue du pont St. Pierre, 31052 Toulouse Cedex, France. E-mail: faye{at}icr.fnclcc.fr

This article has been cited by other articles:

				M. Simonen, Y. Ibig-Rehm, G. Hofmann, J. Zimmermann, G. Albrecht, M. Magnier, V. Heidinger, and D. Gabriel High-Content Assay to Study Protein Prenylation J Biomol Screen, July 1, 2008; 13(6): 456 - 467. [Abstract] [PDF]